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Caracterización de los procesos de adaptación de

hongos formadores de micorrizas a ambientes


afectados por la sequía y salinidad y su implicación en
la mejora de la tolerancia de las plantas a tales
ambientes

Beatriz Estrada Velasco

TESIS DOCTORAL

Granada, 2012
Editor: Editorial de la Universidad de Granada
Autor: Beatriz Estrada Velasco
D.L.: GR 1058-2013
ISBN: 978-84-9028-489-6
Characterization of the adaptation of mycorrhizal
fungi to environmnets affected by drought and salinity
and their implications in the improvement of plant
tolerance to such environments

Beatriz Estrada Velasco

DOCTORAL THESIS

Granada, 2012
UNIVERSIDAD DE GRANADA
FACULTAD DE CIENCIAS
Departamento de Fisiología Vegetal

CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS


ESTACIÓN EXPERIMENTAL DEL ZAIDÍN
Departamento de Microbiología del Suelo y Sistemas Simbióticos

Memoria presentada para aspirar al grado de Doctor en Biología


(con la mención “Doctor Internacional”)

Fdo.: Beatriz Estrada Velasco


Licenciada en Ciencias Biológicas por la Universidad Autónoma de Madrid

Granada a 14 de Diciembre de 2012

VºBº Directores de la Tesis

Fdo.: José-Miguel Barea Navarro Fdo.: Juan Manuel Ruiz-Lozano


Profesor de Investigación del CSIC Investigador Científico del CSIC
Estación Experimental del Zaidín Estación Experimental del Zaidín
Esta Tesis Doctoral ha sido realizada en el Departamento de
Microbiología del Suelo y Sistemas Simbióticos de la Estación
Experimental del Zaidín del Consejo Superior de
Investigaciones Científicas (CSIC) de Granada en el seno del
grupo de investigación de micorrizas.

Este trabajo ha sido financiado mediante una beca predoctoral


concedida por dos Proyectos de Excelencia de la Junta de
Andalucía, P06-CVI-01876 de mismo nombre que la presente
Tesis y P11-CVI-7107 titulado “Regulacion por micorrizas
arbusculares de la respuesta fisiológica integrada a la salinidad
en plantas de arroz”.
A mis padres
y hemano
ÍNDICE
NATURALEZA, INTERÉS Y OBJETIVOS 3
INTEREST OF THE STUDY AND AIMS 7
INTRODUCCIÓN 11
1. Ecosistemas Mediterráneos: Parque Natural Cabo de Gata 13
1.1. Parque Natural Cabo de Gata: interés y problemática. 15
2. El estrés salino 17
2.1. Efecto de la salinidad en las plantas 18
2.2. Sistemas implicados en la tolerancia a la salinidad en plantas 20
3. Las Micorrizas 21
3.1. Las Micorrizas Arbusculares 23
3.2. Clasificación y filogenia de los hongos MA 24
3.3. Ciclo de vida 26
3.4. Significado de los hongos MA en el sistema suelo-planta 28
4. Efecto de la salinidad en las micorrizas arbusculares 29
5. Simbiosis MA y tolerancia al estrés salino en plantas 30
6. Bibliografía 32

CAPÍTULO 1: Diversidad de hongos micorrícico arbusculares en la


rizosfera de Asteriscus maritimus (L), una planta representativa de
ecosistemas Mediterráneos áridos y salinos 45

Resumen

CHAPTER 1: Diversity of arbuscular mycorrhizal fungi in the rhizosphere


of Asteriscus maritimus (L), a representative plant species in arid and saline
Mediterranean ecosystems 47

Abstract 48
Introduction 48
Material and Methods 50
Results 52
Discussion 56
Conclusions 59
References 60
CAPÍTULO 2: Diversispora clara (Glomeromycetes) – una nueva especie de
dunas salinas del Parque Natural Cabo de Gata (España) 69

Resumen

CHAPTER 2: Diversispora clara (Glomeromycetes) – a new species from


saline dunes in the Natural Park Cabo de Gata (Spain) 71

Abstract 72
Introduction 72
Material and Methods 72
Results 74
Discussion 76
References 79

CAPÍTULO 3: Un aislado de Glomus intraradices nativo de un área


Mediterránea salina muestra tolerancia a la salinidad y elevada eficiencia
simbiótica en plantas de maíz bajo condiciones de estrés salino 85

Resumen

CHAPTER 3: A native Glomus intraradices strain from a Mediterranean


saline area exhibits salt tolerance and enhanced symbiotic efficiency with
maize plants under salt stress conditions 87

Abstract 88
Introduction 89
Material and Methods 91
Results 97
Discussion 105
References 110

CAPÍTULO 4: Hongos micorrícico arbusculares nativos de áreas salinas


Mediterráneas aumentan la tolerancia de plantas de maíz mediante la
mejora de la homeostasis iónica 121

Resumen
CHAPTER 4: Arbuscular mycorrhizal fungi native from a Mediterranean
saline area enhance maize plants tolerance to salinity through improved ion
homeostasis 123

Abstract 124
Introduction 124
Material and Methods 127
Results 131
Discussion 139
References 143

CAPÍTULO 5: Hongos formadores de micorrizas arbusculares aislados de


hábitat salinos mejoran el sistema antioxidante del maíz y su tolerancia a la
salinidad 151

Resumen

CHAPTER 5: Native arbuscular mycorrhizal fungi isolated from a saline


habitat improved maize antioxidant systems and plant tolerance to salinity
153

Abstract 154
Introduction 154
Material and Methods 156
Results 161
Discussion 167
References 170

CAPÍTULO 6: Importancia de la inoculación con hongos nativos


formadores de micorrizas arbusculares en la especie halófita Asteriscus
maritimus para su completo establecimiento y crecimiento bajo condiciones
de salinidad 179

Resumen
CHAPTER 6 Importance of native arbuscular mycorrhizal inoculation in
the halophyte Asteriscus maritimus for successful establishment and growth
under saline conditions 181

Abstract 182
Introduction 182
Material and Methods 184
Results 190
Discussion 198
References 202

CONCLUSIONES 211
CONCLUSIONS 213
NATURALEZA, INTERÉS Y OBJETIVOS
INTEREST OF THE STUDY AND AIMS
Naturalza, interés y objetivos

NATURALEZA, INTERÉS Y OBJETIVOS

La importancia y singularidad ecológica de los ecosistemas mediterráneos


adquieren especial relevancia en el territorio español. La conjunción de influencias
paleoárticas y paleotropicales han otorgado a la Cuenca Mediterránea una excepcional
riqueza vegetal, entre otras, con un elevado nivel de biodiversidad y un número
considerable de endemismos. Sin embargo aunque estos ecosistemas sean
relativamente estables en ausencia de perturbaciones, poseen una gran fragilidad y baja
resiliencia, lo que se traduce en una limitada capacidad de responder a las alteraciones
del sistema.

Por sus características geomorfológicas y climáticas, así como por la


intervención humana, que en zonas concretas y en periodos históricos definidos ha sido
decisiva, los procesos de degradación del suelo y de la cubierta vegetal en los
ecosistemas en el área mediterránea adquieren unas connotaciones especiales. Estos son
los principales obstáculos para la supervivencia y reproducción de las especies vegetales
que se encuentran en zonas áridas y semiáridas. España es el país europeo con más
extensión de zonas con riesgo de desertificación ocupando las zonas áridas y semi-
áridas casi la mitad del territorio nacional.

El estrés se debe fundamentalmente a condicionantes climáticos (marcada


estacionalidad y coincidencia de estación cálida con estación seca) que produce
patrones de lluvia altamente impredecibles que pueden derivar en una limitación
hídrica, suelos pobres en nutrientes (nitrógeno y fósforo) con alta variación espacial y
temporal y suelos con alta salinidad. El problema de la salinidad se acentúa porque las
escasas lluvias anuales son insuficientes para transportar las sales solubles fuera de la
zona radical de las plantas. A su vez, los altos niveles de evaporación y radiación solar,
característicos de estas regiones, contribuyen a incrementar más la salinidad de los
suelos.

Las plantas han tenido que desarrollar diversos mecanismos fisiológicos y


moleculares para tolerar este tipo de estrés osmótico, como es la salinidad y garantizar
el éxito de su adaptación en este ambiente. El conocimiento de tales mecanismos es
determinante para poder explotar su potencial uso en programas de restauración y de
agricultura asistida.

Una de las estrategias que han desarrollado las plantas para tolerar el estrés salino
se basa en su asociación con microorganismos rizosféricos (hongos y bacterias). En
particular, un componente clave de la microbiota del suelo, son los hongos formadores
de micorrizas arbusculares (MA), que se postulan como uno de los factores más
influyentes en el mantenimiento de la variabilidad, estabilidad, diversidad y

3
Naturaleza, interés y objetivos

productividad de la cubierta vegetal. La raíces MA exploran mayores volúmenes de


suelo, a mayores profundidades y distancias de lo que lo hacen las raíces de las plantas
no micorrizadas, para suministrar agua y nutrientes a sus asociados vegetales. Sin duda,
los ecosistemas mediterráneos y las peculiares especies de plantas que en ellos se
desarrollan, ofrecen un modelo de interés prioritario para desarrollar investigaciones y
estudiar el impacto de las asociaciones micorrícicas en el mantenimiento y desarrollo de
dichos ecosistemas.

Como es bien conocido, las micorrizas, simbiosis hongo-raíz presente en todos


los biomas y ecosistemas terrestres desde hace más de 400 millones de años, son
fundamentales para que las plantas adquieran nutrientes minerales y agua del suelo,
particularmente en condiciones limitantes, así como una mayor resistencia a los estreses
ambientales. Se ha demostrado que la mayoría de las plantas dependen de estar
micorrizadas para establecerse y prosperar. Más del 80% de las especies de plantas
existentes en el Planeta forman simbiosis con micorrizas arbusculares, entre ellas las
propias de ecosistemas mediterráneos. El establecimiento de la micorriza no sólo resulta
ventajoso para la planta y el hongo, sino que también favorece al ecosistema en su
conjunto, ya que mejora la calidad del suelo así como el desarrollo, la diversidad y la
productividad de la cubierta vegetal.

La vegetación de los ecosistemas áridos y semiáridos, además de soportar


condiciones adversas como largos periodos de sequía, intensas temperaturas y
evaporación, a veces se asienta sobre suelos con alto contenido de sales, arenosos con
alto grado de erosión, y con bajos niveles de nutrientes y de agua, entre los factores
principales; lleva a pensar que las MA son un factor que permite a las plantas resistir
estas condiciones adversas. Concretamente, la salinidad es uno de los principales
factores abióticos que limitan la producción vegetal en muchas áreas del mundo que
afecta tanto a plantas como a los microorganismos de la rizosfera, que a su vez,
desarrollan mecanismos de adaptación a dicho estrés. Actualmente hay un creciente
interés por conocer los mecanismos de tolerancia de las plantas frente al estrés salino y
la implicación de los hongos MA a tal tolerancia.

Por todo lo anterior expuesto, el estudio de las MA de ecosistemas áridos y


semi-áridos y salinizados es fundamental porque albergan importantes fuentes de
inóculos de hongos MA, adaptados a esas condiciones. Los hongos MA aislados en los
ecosistemas mediterráneos afectados por problemas de salinidad pueden ser utilizados
como inóculo de plantas para lograr su establecimiento en condiciones naturales de
estrés osmótico, siendo especialmente útiles en prácticas de restauración ambiental de
ecosistemas degradados o en proceso de desertificación.

4
Naturalza, interés y objetivos

En el contexto de los aspectos conceptuales anteriormente expuestos se diseñó


esta Tesis Doctoral, la cual se integra en un proyecto de investigación encuadrado en el
Programa de Proyectos de Excelencia en I+D+i del PIDI (Junta de Andalucía). Aunque
la estrategia de I+D+i que se propone es extrapolable a cualquier ecosistema afectado
por sequía/salinidad, la propuesta se centra en un escenario de estudio definido,
científicamente representativo y socioculturalmente emblemático de Andalucía, como
es el Parque Natural de Cabo de Gata, Almería, donde son características sus largas
cintas arenosas, salinas, dunas y aridez, con una vegetación caracterizada por su
adaptación al territorio así como los hongos MA. Por todo ello, el objetivo general de la
Tesis fue el Investigar la diversidad natural de hongos MA en el ecosistema, así como
los mecanismos (fisiológicos y moleculares) que rigen los procesos de adaptación de
dichos hongos a la salinidad y su percusión en la tolerancia de las plantas
(supervivencia, desarrollo y productividad) bajo condiciones de salinidad.
La consecución de este objetivo general implicó el planteamiento de los siguientes
objetivos específicos:

1. Analizar la diversidad de hongos MA presentes en la rizosfera de las plantas de


dunas y saladares del Parque y establecer un banco de germoplasma de hongos
adaptados al ecosistema.

2. Estudiar los mecanismos de adaptación/tolerancia de los hongos MA a la


salinidad.

3. Determinar los mecanismos fisiológicos por lo que los hongos MA nativos del
Parque incrementan la tolerancia al estrés salino de plantas glicófitas de interés
agronómico y halófitas de interés en revegetación.

Aunque los objetivos propuestos pretenden avanzar en el conocimiento de la ecología y


funcionamiento fisiológico en la relación hongo-planta de los hongos micorrícicos en
ecosistemas semiáridos característicos de ambientes mediterráneos, la investigación
propuesta presenta también un marcado carácter finalista. Este viene determinado por la
posibilidad de evaluar el efecto de la micorrización dirigida sobre el desarrollo de
plantas tanto para restauración de áreas degradadas como para mejora agrícola en áreas
afectadas por problemas de salinidad.

5
Interest of the study and aims

INTEREST OF THE STUDY AND AIMS

The ecological importance and singularity of Mediterranean ecosystems are


particularly relevant in the Spanish territory. The combination of paleotropicals and
paleoartics influences has given the Mediterranean area exceptionally rich vegetation,
among others, with a high level of biodiversity and a significant number of endemic
species. However, although these ecosystems are relatively stable in the absence of
disturbances, they have high resilience and low fragility, resulting in a limited ability to
respond to disturbances in the system

Due to geomorphological and climatic characteristics, as well as human


intervention in specific areas, land degradation and vegetation cover in the
Mediterranean ecosystems acquires special connotations. These are the main obstacles
for the survival and reproduction of plant species found in arid and semiarid areas.
Spain is the European country with the greatest extent of areas at risk of desertification
where the arid and semi-arid areas occupy nearly half of the national territory.

The stress is mainly due to climatic conditions (seasonality with hot and dry
season together) that produces highly unpredictable rainfall patterns that can lead to a
hydric limitation, soils poor in nutrients (nitrogen and phosphorus) and soils with high
salinity. The salinity problem is accentuated by the limited annual rainfall which is
insufficient to transport the soluble salts out of the rhizosphere of the plants. Moreover,
the characteristics of these regions: high levels of evaporation and solar radiation,
contribute to further increase the salinity of soils.

Plants have developed various physiological and molecular mechanisms to


tolerate this type of osmotic stress, such as salinity to ensure the success of adaptation in
this environment. The knowledge of such mechanisms is crucial in order to exploit their
potential use in restoration programs and assisted agriculture.

One strategy that plants have developed to tolerate salt stress is based on its
association with rhizospheric microorganisms (fungi and bacteria). In particular, a key
component of soil microbiota, the arbuscular mycorrhizal fungi (AMF), are postulated
as one of the most influential factors in the maintenance of variability, stability,
diversity and productivity of the vegetation cover. The AM roots explore greater
volumes of soil at greater depths and distances to supply water and nutrients to the host
plant than roots of non-mycorrhizal plants. In fact, Mediterranean ecosystems together
with their particular plant species, offer a model of interest to conduct research projects
to study the impact of mycorrhizal associations in the maintenance and development of
these ecosystems.

7
Interest of the study and aims

As is well known, the mycorrhizal fungus-root symbiosis present in all biomes


and terrestrial ecosystems for over 400 million years, and they are essential for plants to
acquire mineral nutrients and water from the soil (particularly under limited conditions)
and greater resistance to environmental stresses. It has been demonstrated that most
plants depend on mycorrhizal symbiosis to establish and develop. Over 80% of the plant
species on Earth form symbiosis with arbuscular mycorrhiza, including those
characteristic of Mediterranean ecosystems. The mycorrhizal establishment not only
benefits the plant and the fungus, but also the whole ecosystem, improving soil quality
and development and the diversity and productivity of the vegetation cover.

Vegetation of arid and semiarid ecosystems, as well as withstand adverse


conditions (long periods of drought, intense temperatures and evaporation), sometimes
grow on soils with high salt content, or sandy with high erosion, or low nutrient and
water levels, among the main factors, suggesting that the AM symbiosis is a key factor
that allows plants to cope with these adverse conditions. Specially, salinity is a major
abiotic factor limiting plant production in many areas of the world, affecting both plants
and microorganisms in the rhizosphere, which in turn, develop adaptation mechanisms
to such stress. Nowadays there is an increasing interest in understanding the
mechanisms of plant tolerance to salt stress and the involvement of AMF in such
tolerance.

For all the above reasons, the study of the AMF of arid and semi-arid salinized
ecosystems is critical because they are important potential sources of AMF inocula
adapted to these conditions. The AMF isolated in Mediterranean ecosystems affected by
salinity might be used as inoculum to achieve plant establishment under natural
conditions of osmotic stress, being especially useful in environmental restoration
practices of degraded ecosystems or under desertification process. The present Doctoral
Thesis was outlined in the context of the conceptual topics above mentioned and it is
part of a research project conducted within the Excellence Project Program in I+D+i of
PIDI (Junta de Andalucía). Although the strategy of I+D+i proposed can be extrapolated
to any ecosystem affected by drought/salinity, the proposal focuses on a defined
scientifically representative sampling area and socioculturally emblematic part of
Andalucía: the Natural Park Cabo de Gata, Almería, with characteristic long sandy
ribbons, salt marshes, dunes and aridity, together with vegetation adapted to the territory
and AMF. Therefore, the aim of the thesis was To investigate the natural diversity of
AMF in the ecosystem, and the physiological and molecular mechanisms that govern
the processes of adaptation of these fungi to salinity and repercussion on plant
tolerance (survival, development and productivity) under salinity conditions.

8
Interest of the study and aims

To achieve this main objective, the following specific objectives were proposed:

1. To analyze the diversity of AMF present in the rhizosphere of the plants in the
dunes and salt marshes of the Park and establish a germplasm bank of AMF
adapted to the ecosystem.

2. To study the mechanisms of adaptation/tolerance of AMF to salinity

3. To determine the physiological mechanisms by which native AMF from the


Park increase salt stress tolerance of glicophyte plants of agronomic interest and
halophyte plants of revegetation interest.

Although the proposed objectives are intended to advance in the knowledge of the
ecology and physiological functioning in the relationship fungus-plant of AMF in arid
ecosystems typical of Mediterranean environments, the research also presents a strong
proposal. The latter is determined by the ability to evaluate the effect of conducted
mycorrhization in the development of both plants for restoration of degraded areas and
agricultural improvement in areas affected by salinity.

9
INTRODUCCIÓN
Introducción

INTRODUCCIÓN

1. Ecosistemas Mediterráneos: Parque Natural Cabo de Gata

Los ecosistemas mediterráneos incluyen un importante número de comunidades bióticas


que se desarrollan bajo la influencia del denominado macrobioclima mediterráneo según
la clasificación bioclimática de (Rivas-Martínez, 1981). Esta unidad tipológica de rango
superior se puede encontrar en cualquier altitud y valor de continentalidad en todos los
territorios extratropicales de la Tierra pertenecientes a las cinturas subtropical y
eutemplada, principalmente entre los 30 y 40º de latitud N y S. Característica
fundamental para catalogar una zona como “de clima mediterráneo” es que en ellas
existen al menos dos meses consecutivos con aridez durante el verano, es decir, en los
que el valor en milímetros de la precipitación media del bimestre más cálido del
trimestre estival es menor del doble de la temperatura media del bimestre más cálido del
trimestre estival expresada en grados centígrados (P <2T). También es una característica
del clima mediterráneo la alternancia de la estación cálida y seca con una estación fría y
húmeda, con precipitaciones irregulares y esporádicas, pero frecuentemente torrenciales
(López-Bermúdez and Albaladejo, 1990). Aunque se le denomine clima mediterráneo,
debido a que una de las zonas más representativas con este clima son las regiones
circundantes del mar Mediterráneo, hay numerosas zonas que comparten las
características de este clima, con mayor representación territorial en el centro y en el
occidente de todos los continentes excepto en la Antártida.

La parte árida y semi-árida de la región mediterránea, en torno a dicho Mar


corresponde a la zona oriental de la Península Ibérica, las costas septentrionales de
África (Argelia, Egipto, Libia, Marruecos y Túnez) y las islas de Creta, Chipre y las
Baleares. En estas áreas, las precipitaciones anuales están por debajo de los 400 mm.
(Wheeler and Kostbade, 1990). Además del clima, tanto el relieve, como el sustrato
litológico, la cubierta vegetal y la intervención humana son factores esenciales que
definen las cualidades y funcionamiento de los ecosistemas mediterráneos. Los suelos
sobre los que se desarrollan los ecosistemas mediterráneos presentan características
funcionales diferentes de los que se encuentran en ecosistemas templados y tropicales.
Son suelos más jóvenes y menos profundos que los tropicales, (debido a tasas de
meteorización de la roca sensiblemente inferiores) y han estado sujetos a una mayor tasa
de erosión que los suelos de ecosistemas templados (debido a unos usos más intensivos
y prolongados por parte del hombre, y quizás por la mayor recurrencia de incendios)
(Gallardo et al., 2009). Estas diferencias imponen las primeras restricciones a las
plantas que se desarrollan sobre estos suelos. La escasa profundidad impone un límite al
tamaño de los individuos, mientras que la erosión de los horizontes superficiales limita
la cantidad de materia orgánica y los nutrientes que de ella se derivan (Yaalon, 1997).

13
Introducción

Los ecosistemas de la región mediterránea son muy variados, y su vegetación


climácica pueden ser bosques esclerófilos de hoja perenne, bosquetes espinosos, estepas
templadas o incluso semidesiertos helados, todos ellos adaptados a soportar un periodo
de aridez de hasta nueve meses. Por ello la vegetación mediterránea desarrolla
principalmente dos tipos de alternativas de adaptación xerófila: la presencia de
estructuras aéreas persistentes (como es el aso de los matorales) y la que expresa la
vegetación de temporada, en el caso de las plantas terófitas, como por ejemplo los
pastizales de especies anuales. Hay que destacar además, que la biomasa subterránea
respecto de la aérea es más alta que en otros ecosistemas (Montalvo, 1992). Otra
característica importante de estos ecosistemas es su excepcional riqueza florística, la
cual ha sido determinada por la conjunción de factores bióticos y ambientales, como son
las variaciones climáticas y los cambios de las posiciones relativas de las grandes masas
continentales (Blanca and Morales, 1991; López-González, 2001). En concreto, en la
cuenca mediterránea de las 25.000 plantas fanerógamas identificadas hasta el momento
(alrededor del 10 % de todas las plantas conocidas en la Tierra), más de la mitad son
endémicas de la región. No es de extrañar que el la cuenca mediterránea se considere
uno de los lugares con mayor biodiversidad del mundo, siendo los endemismos un
componente integral y fundamental (Thompson et al., 2005).

En las áreas mediterráneas, las lluvias escasas e irregulares, con largos, secos y
calurosos veranos, junto con la presión antropogénica y abandono de suelos agrícolas,
son factores determinantes de la degradación del ecosistema (Barea et al., 2006). En
particular en el sureste de España, esta situación de estrés múltiple está llevando a
desertificación de parte del territorio (Francis and Thornes, 1990; Albaladejo et al.,
1996). donde aproximadamente un 18% del territorio presenta graves procesos de
erosión y desertificación, siendo la cifra en Andalucía del 36%, debido principalmente a
las características accidentales del relieve, con fuertes pendientes y desniveles, que
acentúan el efecto de las precipitaciones (Cornejo, 2006).

En general, es difícil pensar una revegetación natural espontánea en áreas donde


las precipitaciones están por debajo de 350 mm (Barea et al., 2006). La franja costera
del Sureste español no es tan sólo la región de aridez más extremada de la Península
ibérica, sino también de toda Europa, por lo que el estudio y conservación de zonas de
especial interés ecológico, como es el Parque Natural de Cabo de Gata elegido como
modelo del presente estudio, son de vital importancia debido a sus características únicas
y endemismos.

14
Introducción

1.1. Parque Natural Cabo de Gata: interés y problemática.

El Parque Natural de Cabo de Gata-Níjar, a través del Decreto 314/1.987 de 23 de


Diciembre, es el primer Parque Natural marítimo-terrestre de Andalucía y el de mayor
superficie y relevancia ecológica de todo el Mediterráneo occidental europeo. Su origen
volcánico, sus salinas, sus dunas fósiles y la importancia ecológica de su flora y su
fauna son algunas de las razones por las que se han protegido 38.000 hectáreas terrestres
y 12.000 hectáreas de costa hasta una milla marítima (Figura 1).

Límites
Límites deldel PN
Reserva terrestre
Reserva
Reserva marina
Reserva

Figura 1. Localización del Parque Natural de Cabo de Gata y sus zonas protegidas

Este enclave es uno de los pocos lugares, en toda Europa, protegido por su carácter
semiárido y estepario. Las condiciones climáticas de sequedad de Cabo de Gata son
semejantes a las que existen en extensos territorios de África del Norte o de Oriente
Medio, lo que identifica este lugar como el enclave más árido de Europa Occidental
(Geiger, 1973). A pesar de ello y de su aparente aspecto desértico encierra formas de
vida animal y vegetal muy peculiares, que han logrado adaptarse a extremas
condiciones de aridez, caracterizada por su elevada insolación media (2960 horas), el
índice de precipitaciones mas bajo de la península y la suavidad de su régimen térmico

15
Introducción

(temperatura media entre 15 y 22ºC, no soliendo descender nunca de los 12ºC). El


reparto anual de precipitaciones queda restringido a una quincena de días ligados a
periodos favorables (otoño-invierno), llegando en ocasiones, a recogerse, en un solo día,
más del 40% de la media anual (Asensio Grima et al., 2005).

El área incluye diferentes tipos de paisajes asociados a materiales volcánicos y


calizas arrecifales, distinguiéndose grandes unidades paisajísticas: Planicie, Valles,
Piedemontes, Lomerios y Montañas. La vegetación es muy variada, con una alta
proporción de endemismos y una alta diversidad de comunidades vegetales. De las más
de 200.000 especies vegetales catalogadas en el ámbito mediterráneo, en el Parque
Natural de Cabo de Gata-Nijar aparecen en torno a un millar, cantidad sorprendente
para espacio tan limitado en extensión y altura con endemismos locales como el
Dragoncillo del Cabo (Antirrhinum charidemi), Clavelinas del Cabo (Dianthus
charidemi), la Zamarrilla del Cabo (Teucrium charidemi) y el Azafrán del Cabo
(Androcymbium europaeum). La vegetación dominante en la zona de montaña está
constituida por matorrales de esparto (Stipa tenacísima), albaida (Anthyllis cytisoides),
tomillo (Thymus vulgaris) y romero (Rosmarinus officinalis) asociados a otras especies
como palmito, (Chamaerops humilis) única palmera autóctona en el continente europeo
y símbolo del Parque, o cornical (Periploca angustifolia), con una gran diversidad
florística y estructural. En las zonas arenosas propias de la Planicie aparecen otras
especies como Tamarix sp. y azufaifo (Ziziphus loti), siendo las formaciones de esta
última las que mayor superficie ocupan en la Península. En las zonas arenosas y
saladares abundan además familias de Quenopodiáceas, Leguminosas y Asteráceas,
siendo una especie perteneciente a esta última familia, Asteriscus maritimus, de alto
interés en la conservación del suelo gracias a su extenso sistema radicular (Rodríguez et
al., 2005). Los pastizales ocupan amplias superficies, asociadas principalmente a
cultivos de secano en zonas de Piedemonte, en la Montaña, o en dunas de la Planicie
(Escribano et al., 2008). Respecto a las comunidades de vertebrados terrestres, cabe
distinguir entre la sierra y zonas esteparias inmediatas y las salinas. En el primer
dominio, según los trabajos previos a la declaración de Parque Natural, existen 54
especies. El valor de su extraordinario biodiversidad ha sido reconocido en 1997 por la
U.N.E.S.C.O con la catalogación de este Parque Natural como Reserva de la Biosfera.
El mundo marino protegido, tiene fondos rocosos y arenosos; la diversidad de su
colonización vegetal alberga más de 1400 elementos vegetales (cabe destacar la
importancia ecológica de los campos de Posidonia oceanica) y animales reconocidos
hasta la fecha (Escribano, 2002).

Los modelos regionales de cambio climático prevén un aumento de la


temperatura, una disminución de la precipitación y un mayor número de eventos
torrenciales en los ecosistemas áridos (IPCC, 2007). Según esto las condiciones de
aridez en el Parque se intensificarán, lo que puede incrementar la erosión y degradación

16
Introducción

del suelo provocando una pérdida de cubierta vegetal y microbiota del suelo (Requena
et al., 1996). La microbiota del suelo juega un papel fundamental en la regulación de los
ecosistemas terrestres, influyendo en la productividad, diversidad y estructura de las
comunidades vegetales (van der Heijden et al., 2008). Entre los organismos que habitan
en el suelo cabe destacar por su función ecológica los hongos micorrícico arbusculares
(ver punto 3). El deterioro de los sistemas suelo-planta, en cuanto que afecta a las
relaciones planta-microorganismos, desencadena un círculo vicioso de efectos
negativos. Si no hay plantas, se degrada la vida microbiota del suelo y si no hay
propágulos, cualquier proceso natural o inducido de revegetación presenta problemas
para prosperar adecuadamente (Marulanda, 2006). Por lo tanto para evitar la
degradación de este tipo de ecosistemas, que por sus condiciones extremas son más
sensibles, es necesario realizar más estudios que desvelen las complejas interacciones
que se establecen en ellos (Martínez and Pugnaire, 2009).

2. El estrés salino

La salinización de los suelos es uno de los mayores problemas no sólo a nivel ecológico
sino también agronómico. A nivel ecológico es la causante de la pérdida de
comunidades naturales de plantas y acelera los procesos de degradación del suelo y
desertificación (Alguacil et al., 2011). La salinidad es además el estrés abiótico que
afecta más negativamente al crecimiento vegetal y por tanto a la producción agrícola,
además de limitar el uso de nuevas áreas potenciales de cultivo. Las plantas pueden
clasificarse en base a su tolerancia a la salinidad como halófitas (tolerantes) y glicófitas
(sensibles), habiendo grandes diferencias dentro de cada grupo en base al nivel de
tolerancia (Greenway and Munns, 1980). Debido a que la mayor parte de los cultivos
son glicófitos, el exceso de sales en el suelo ocasiona pérdidas importantísimas en las
explotaciones agrícolas (Pitman and Läuchli, 2002). Los factores ambientales están
estrechamente relacionados con la distribución de las zonas afectadas por salinidad. Por
tanto en los climas áridos y semiáridos, caracterizados por escasez de lluvias,
temperaturas extremas y alta velocidad de evaporación (Brito et al., 2011), el principal
factor limitante de la fertilidad de los suelos y la productividad de los cultivos lo
constituye la salinidad (Evelin et al., 2009). A nivel mundial hay más de 800 millones
de hectáreas afectadas por la salinidad, más del 6% del área total de la superficie
terrestre (Munns and Tester, 2008). En particular, en la cuenca mediterránea alrededor
de 16 millones de hectáreas están afectadas por la salinidad y en la España la cifra
asciende hasta 840.000 hectáreas (Serrano, 2009). Además de las causas naturales, la
actividad humana, en concreto las malas prácticas de cultivo y el riego, han contribuido
al incremento alarmante de sales en el suelo (Mahajan and Tuteja, 2005).

17
Introducción

2.1. Efecto de la salinidad en las plantas

La salinidad del suelo tiene un efecto negativo en el establecimiento, crecimiento y


desarrollo de las plantas (Evelin et al., 2009). Las plantas que crecen en áreas afectadas
por salinidad son sometidas a tres estreses fisiológicos: toxicidad iónica, estrés osmótico
y desequilibrio nutricional (Munns and Tester, 2008).

• Toxicidad iónica: el exceso de sales conduce a una acumulación excesiva de


Na+ y Cl- en el citosol de la célula. Aunque el Cl- es un micronutriente esencial
para las plantas implicado en la regulación de importantes funciones celulares
como actividades enzimáticas, potencial de membrana, co-factor en la
fotosíntesis y gradientes de pH (Marschner, 1995; White and Broadley, 2001),
también puede ser tóxico en grandes concentraciones, siendo el anión más
abundante en suelos salinos (Xu et al., 2000). Sin embargo, la toxicidad iónica
es producida principalmente por el Na+. El Na+ compite con el K+ para unirse a
sitios esenciales debido a la similitud en sus propiedades fisicoquímicas aunque
no puede sustituir sus funciones metabólicas en el citoplasma (Tester and
Davenport, 2003). El K+ es fundamental para el mantenimiento homeostático en
el citosol en plantas afectadas por estrés salino (Zhu, 2003). La alteración que
provoca el Na+ en la captación de K+ altera la relación K+/Na+ de la célula, que
es incluso más importante que la concentración absoluta de Na+ (Demidchik and
Maathuis, 2007). El K+ es un ión esencial para la fotosíntesis, la síntesis de
proteínas, activación de multitud de enzimas y juega un papel fundamental en el
ajuste osmótico, mantenimiento de la turgencia y en los procesos estomáticos
(Maathuis and Amtmann, 1999). Por tanto el exceso de Na+ afecta a la estructura
de enzimas y otras macromoléculas, daña orgánulos celulares, perturba el
proceso de fotosíntesis y respiración, inhibe la síntesis de proteínas e induce
deficiencias iónicas.

• Estrés osmótico: la salinidad del suelo dificulta la extracción de agua por parte
de las raíces ya que son expuestas a un bajo potencial osmótico. Esta
disminución de la capacidad de las plantas de absorber agua del suelo supone
una reducción de la expansión foliar y una pérdida de turgencia, siendo más
evidente la reducción del crecimiento en la parte aérea que en las raíces (Munns,
2002). Por tanto las plantas son expuestas a sequía fisiológica y tienen que
mantener bajo el potencial osmótico interno ya que a altas concentraciones de
Na+ en el medio extracelular las plantas deben evitar que el agua salga de las
raíces al suelo (Ruiz-Lozano et al., 2012). Para mantener el potencial osmótico
interno y la actividad citosólica el agua ha de pasar de la vacuola al citosol. Esto
conlleva una reducción en el turgor, en la expansión celular, en la velocidad de
división celular y afecta al desarrollo reproductivo (Munns and Tester, 2008).

18
Introducción

• Desequilibrio nutricional: en suelos con exceso de sales el incremento de los


contenidos celulares de Na+ y Cl- interfiere en la disponibilidad, captación,
transporte y distribución no sólo de agua sino de otros nutrientes minerales
necesarios para la planta como son, entre otros, K+, Ca2+ y NO3- (Marschner,
1995; Tuteja, 2007). El Na+ afecta directamente en la toma de otros nutrientes
debido a la interferencia que produce en la actividad de transporte iónico a nivel
de la membrana celular de la raíz. Este efecto es más evidente en los
transportadores y canales de K+, afectando tanto la toma de este macronutriente
como a su homeostasis. Además, cuando el Na+ atraviesa la membrana
plasmática se observa una significativa despolarización del potencial eléctrico de
la misma (Shabala and Cuin, 2008). Esta despolarización dificulta la entrada de
K+ en la célula y aumenta su salida.

Además de los tres estreses fisiológicos mencionados, la salinidad también induce la


producción de especies reactivas de oxígeno (abreviado ROS, por las singlas en inglés
de Reactive Oxygen Species), generando daño oxidativo en las plantas (Ding et al.,
2010). El exceso de ROS puede dañar la estructura de las enzimas y otras
macromoléculas de las células vegetales (Mittler, 2002). De hecho, la salinidad reduce
la disponibilidad de CO2 atmosférico debido al incremento del cierre de los estomas y el
consumo de NADPH en el Ciclo de Calvin también se ve afectado. Cuando la
ferredoxina se sobre-reduce durante la transferencia electrónica durante la fotosíntesis,
los electrones excedentes deben ser transferidos desde el fotosistema I al oxígeno para
formar el radical superóxido (O2•−) en la llamada Reacción de Mehler, que inicia una
reacción en cadena que produce más radicales de oxígeno perjudiciales (Mittler, 2002).
Estos incluyen el oxígeno singlete (1O2), peróxido de hidrógeno (H2O2) y radicales
hidroxilo (OH•). Estas ROS son citotóxicas y pueden destruir el metabolismo normal de
las células produciendo daño oxidativo de lípidos, proteínas y ácidos nucleicos cuando
son producidas en exceso (Miller et al., 2010). La peroxidación lipídica provoca
cambios en la estructura y función de las membranas, altera la compartimentación de
iones y la pérdida de potencial eléctrico, inhibe el transporte de metabolitos, modifica
los receptores de hormonas y la producción de mensajeros químicos (Nandwal et al.,
2007). A nivel de proteínas, las ROS pueden fragmentarlas o formar uniones entre ellas
ya que actúan a nivel de las cadenas laterales de los residuos aminoacídicos (Therond et
al., 2000). El estrés oxidativo puede ocasionar también la alteración y ruptura de
cadenas de ADN, originando mutaciones (Moller et al., 2007). Por tanto hay una
necesidad constante de mecanismos que mantengan las ROS bajo niveles no
perjudiciales para las plantas ya que la acumulación de ROS depende en gran medida
del balance entre su producción y retirada (Mittler et al., 2004).

19
Introducción

2.2. Sistemas implicados en la tolerancia a la salinidad en plantas

Debido al efecto negativo que produce el estrés salino en las plantas, éstas han
desarrollado mecanismos bioquímicos y moleculares para paliar el efecto negativo de la
salinidad (Figura 2). Para la mayoría de las plantas, el mecanismo para conferir mayor
tolerancia a la salinidad es restringir la absorción de Na+ y Cl- y mantener la del resto de
macronutrientes a niveles normales (Teakle and Tyerman, 2010).

s Toxicidad iónica Inhibición del


se s
s tre g i c o establecimiento,
e ló Estrés osmótico
io crecimiento y
fi s
Desequilibrio desarrollo
nutricional Daño oxidativo
Estrés
salino
re Homeostasis iónica
sp
ue
s ta Control y
Ajuste osmótico Tolerancia
reparación
Sistemas de daños
antioxidantes

Figura 2. Efectos y respuesta del estrés salino en plantas

• Homeostasis iónica: además de la disminución en la toma de sales a través de la


raíz, es necesario su partición a nivel celular y de tejido para que no formen
concentraciones tóxicas en el citosol de las hojas durante la transpiración
(Munns, 2005). También es necesaria la extrusión de iones en el apoplasto y el
secuestro de iones en compartimentos intracelulares y tejidos menos sensibles
(Flowers and Colmer, 2008). Para ello las plantas utilizan mecanismos que
disminuyen la concentración de Na+ en el citoplasma (especies excluidoras) y
mecanismos que disminuyen la concentración en el citoplasma (especies
incluidoras). Para limitar la entrada de Na+ en la raíz, transporte y distribución
hacia las hojas y su compartimentación en vacuolas, las plantas han desarrollado
diversas estrategias. Esto se consigue fundamentalmente mediante sistemas de
transporte de iones a través de las diferentes membranas celulares (Zhu, 2003).
Los progresos en genética molecular y fisiología de las plantas apuntan como
mecanismo clave en la tolerancia al estrés salino la homeostasis del Na+ y del
K+, el cual es un proceso complejo y coordinado que conlleva el mantenimiento
en el citoplasma de una alta razón K+/Na+ (Tester and Davenport, 2003).

20
Introducción

• Ajuste osmótico: el ajuste osmótico permite evitar la pérdida de agua


favoreciendo a las células vegetales mantener el turgor y los procesos que
dependen de él como la expansión celular y crecimiento, la apertura de estomas
y fotosíntesis, además de mantener un gradiente de potencial de agua favorable
para que entre el agua en la planta. El balance osmótico en el citoplasma se
consigue mediante la acumulación de osmolitos compatibles, es decir, solutos
que no inhiban procesos metabólicos (Hasegawa et al., 2000). Los solutos que
participan en el ajuste osmótico pueden ser iones inorgánicos, principalmente
K+, o compuestos orgánicos como azúcares (principalmente glucosa y fructosa,
pero también trehalosa, rafinosa, fructanos), polialcoholes (glicerol, inositoles
metilados), metabolitos cargados (glicina betaína) y aminoácidos como la
prolina (Flowers and Colmer, 2008). El coste energético que requiere la síntesis
de osmolitos orgánicos es en parte responsable de la reducción del crecimiento.

• Sistemas antioxidantes: actualmente se está avanzando en el conocimiento de


las ROS producidas en plantas como moléculas asociadas a la transducción de
señales que se desencadenan ante situaciones de estrés (Foyer and Noctor,
2009). Bajo condiciones normales, las ROS son producidas en bajos niveles en
orgánulos como cloroplastos, la mitocondria o los peroxisomas. Sin embargo
durante el estrés salino, la tasa de producción de ROS se ve dramáticamente
incrementada debido a la acumulación de NADPH y ATP que no son
consumidos. Por tanto son necesarios mecanismos de protección y reparación de
los daños producidos por el exceso de ROS en condiciones de estrés. Para ello
las plantas han desarrollado sistemas eficientes de secuestración de ROS, que
incluyen tanto enzimas antioxidantes (localizadas en diferentes compartimentos
celulares) como pequeñas moléculas no enzimáticas que funcionan directamente
como sistema de defensa antioxidante o indirectamente como cofactores de
distintas actividades enzymáticas (Scheibe and Beck, 2011).

3. Las Micorrizas

La mayoría de las plantas que crecen sobre la corteza terrestre viven asociadas, en
forma de simbiosis mutualista, con ciertos hongos del suelo, constituyendo las llamadas
micorrizas, término que deriva del griego mykos (hongo) y riza (raíz). Este término fue
utilizado por primera vez por el botánico alemán A.B. Frank a finales del siglo XIX
(Frank, 1885), haciendo referencia a la simbiosis observada entre las raíces de ciertos
árboles con un micelio fúngico a la que ya, en aquel momento, supuso la función de

21
Introducción

favorecer las condiciones hídricas de la zona adyacente al sistema radical, además de un


importante papel nutricional.

Sin embargo, no fue hasta los años 50 cuando se comenzó a poner de manifiesto
la importancia real y el significado de estas asociaciones, así como su presencia en la
práctica totalidad de los sistemas suelo-planta (Barea and Jeffries, 1995). El hongo,
habitante común de los suelos, contacta las raíces y coloniza biotróficamente la corteza
de las mismas, sin causar daño a la planta y sin llegar a activar por completo su reacción
de defensa (Smith and Read, 2008). Una vez que el hongo ha colonizado la raíz,
desarrolla un micelio externo que coloniza el suelo que rodea la raíz y ayuda a la planta
a adquirir nutrientes minerales y agua, además de conferirle una mayor resistencia a los
estreses ambientales (Barea et al., 2012). Según esto, en la mayoría de los casos, el
órgano de captación de nutrientes de la planta sería la micorriza y no la raíz
propiamente dicha (Harley and Smith, 1983). A su vez la planta hospedadora
proporciona al hongo simbionte (heterótrofo) compuestos carbonados procedentes de la
fotosíntesis, así como un nicho ecológico protegido donde poder completar su ciclo de
vida (Brundrett, 2004).

Un aspecto importante de las micorrizas es su universalidad ya que se


encuentran prácticamente en todos los ecosistemas terrestres (Allen, 1991).
Actualmente se estima que el 90% de las plantas que crecen sobre la Tierra están
micorrizadas (Smith and Read, 2008). Sólo en unas pocas familias botánicas hay
especies que no forman micorrizas, tal es el caso de las crucíferas, quenopodiáceas y
ciperáceas (Barea and Honrubia, 1993). Las plantas y sus micorrizas han co-
evolucionado desde hace 400-460 millones de años según registros fósiles (Fósil
Rhynie, datado en 370 millones de años) y estudios sobre filogenia y evolución
(Redecker et al., 2000).

A pesar de muchas similitudes en cuanto a función y, en algunos casos


morfología, se pueden reconocer cinco tipos de micorrizas en base a las estructuras
formadas y a la naturaleza de los simbiontes implicados (Barea, 1998). Estos cinco tipos
de micorrizas se encuadran en 3 grupos tróficos: ectotróficas, ectendotróficas y
endotróficas. Este último grupo trófico está formado por las endomicorrizas ericoides,
orquidoides y arbusculares. El objeto de esta investigación se centra en este último
grupo: los “hongos formadores de micorrizas arbusculares”, comúnmente llamadas por
el acrónimo MA (Smith and Read, 1997).

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Introducción

3.1. Las Micorrizas Arbusculares

Las micorrizas arbusculares (MA) constituyen el tipo de simbiosis más extendido en la


naturaleza. Se estima que está presente en el 80-85% de las plantas, encontrándose en la
práctica totalidad de las especies de interés agronómico e industrial (Jeffries and Barea,
2012). La principal característica morfológica de estos hongos son los arbúsculos,
estructuras típicas de la colonización que el hongo desarrolla en el interior de las células
de la corteza de la raíz por ramificación dicotómica repetida de sus hifas.
El largo periodo de vida en común de estos hongos y plantas simbiontes ha
condicionado el elevado grado de mutualismo y dependencia que los simbiontes
muestran entre sí. De hecho, la mayoría de las plantas son “micotróficas”, necesitan
estar micorrizadas para prosperar, mientras que el hongo MA es un “simbionte
obligado”(Barea and Azcón-Aguilar, 2012) Esta es una de las principales limitantes del
estudio de este grupo de hongos, ya que no es posible su multiplicación en condiciones
axénicas (Bago and Cano, 2005).

Las bases fundamentales sobre las que se establece la simbiosis son nutritivas:
los hongos MA reciben fotosintatos de la planta a cambio de una mejora en la toma de
nutrientes, fundamentalmente fósforo, que es el elemento limitante en el ecosistema
terrestre (Bucher, 2007), pero también potasio, calcio, magnesio, azufre, hierro, zinc,
cobre, manganeso (Boomsma and Vyn, 2008), así como agua del suelo, dada la mayor
accesibilidad del micelio externo del hongo a recursos del suelo más distantes del
sistema radical (Ferrol and Pérez-Tienda, 2009). No obstante, la asociación genera otros
beneficios, entre los que destacan una mayor resistencia de la planta micorrizada al
ataque de patógenos del sistema radical (Hooker et al., 1994; Pozo et al., 2009), a la
presencia de metales pesados en el suelo (del Val et al., 1999; González-Guerrero et al.,
2009), al estrés hídrico (Augé, 2001; Ruíz-Lozano and Aroca, 2010) o a condiciones
extremas de pH del suelo (Clark et al., 1999; Oliveira et al., 2005; Cornejo et al., 2008).
Además, la red de hifas extrarradicales y ciertas sustancias secretadas por el hongo
durante la asociación favorecen la formación de agregados estables en el suelo y, por
tanto, la conservación de la estructura física del mismo (Wright and Upadhyaya, 1998;
Jeffries and Barea, 2012). Es por esto que las MA desempeñan un papel fundamental en
la supervivencia y desarrollo de las plantas, sobre todo, en suelos sometidos a
condiciones de estrés (sequía, salinidad, cambios bruscos de temperatura, deficiencia de
nutrientes), como los que caracterizan a los ecosistemas mediterráneos (Requena et al.,
2001; Sánchez-Castro et al., 2012).

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Introducción

3.2. Clasificación y filogenia de los hongos MA

Los primeros intentos de clasificar los hongos MA datan de finales del siglo XIX y
comienzos del XX y se basaron exclusivamente en criterios morfológicos referenciados
en las esporas. En estos estudios estos hongos se incluyeron en la familia Endogonaceae
dentro del phylum Zygomycota (Gerdemann and Trappe, 1974). En los últimos años la
sistemática del grupo de hongos formadores de MA ha sufrido varias modificaciones,
sobre todo por la incorporación de técnicas moleculares en el estudio de la filogenia de
estos hongos. Actualmente se ha demostrado la naturaleza monofilética de este grupo de
hongos mediante el análisis de 18S ADNr. Esto ha permitido incluirlos en un nuevo
phylum, denominado Glomeromycota (Schüβler et al., 2001). En la actualidad este
phylum es motivo de debate y posee dos posibles clasificaciones que se muestran a
continuación (Tablas 1 y 2).

Tabla 1. Clasificación de los hongos MA según Krüger et al. 2012.


CLASE ORDEN FAMILIA GÉNERO
Glomeromycetes Glomerales Glomeraceae Glomus
Funneliformis
Septoglomus
Rhizophagus
Sclerocystis
Claroideoglomeraceae Claroideoglomus
Diversisporales Diversisporaceae Diversispora
Redeckera
Pacisporaceae Pacispora
Acaulosporaceae Acaulospora
Gigasporaceae Scutellospora
Gigaspora
Racocetra
Archaeosporales Ambisporaceae Ambispora
Archaeosporaceae Archaeospora
Geosiphonaceae Geosiphon
Paraglomerales Paraglomeraceae Paraglomus

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Introducción

Tabla 2. Clasificación de los hongos MA según Oehl et al. 2011.


CLASE ORDEN FAMILIA GÉNERO
Glomeromycetes Glomerales Glomeraceae Glomus
Funneliformis
Septoglomus
Simiglomus
Entrophosporaceae Claroideoglomus
Albahypha
Viscospora
Entrophospora
Diversisporales Diversisporaceae Diversispora
Redeckera
Otospora
Tricispora
Sacculosporaceae Sacculospora
Pacisporaceae Pacispora
Acaulosporaceae Acaulospora
Kuklospora
Gigasporales Scutellosporaceae Scutellospora
Orbispora
Dentiscutataceae Fuscutata
Dentiscutata
Quatunica
Racocetraceae Cetraspora
Racocetra
Gigasporaceae Gigaspora

Archaeosporomycetes Archaeosporales Ambisporaceae Ambispora


Archaeosporaceae Archaeospora
Intraspora
Geosiphonaceae Geosiphon

Paraglomeromycetes Paraglomerales Paraglomeraceae Paraglomus

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3.3. Ciclo de vida

En los hongos MA no se ha demostrado una reproducción sexual reconocida sino que


producen esporas de resistencia multinucleadas, pudiendo llegar hasta los 20.000
núcleos por espora (Ferrol et al., 2004). El ciclo de vida se inicia partiendo de los
propágulos de estos hongos, que se mantienen en el suelo en forma de esporas, redes de
micelio, o colonizando raíces activas o fragmentos de éstas que permanecen en el suelo
(Sánchez-Castro, 2009) La figura 3 resume el ciclo de vida de las micorrizas
arbusculares.

Apresorio

Esporas

Extrarradical

Intrarradical BAS-espora

Arbúsculo Branched Absorbing


Structure (BAS)

Figura 3. Ciclo de vida de los hongos formadores de MA (Tomado de Porcel et al. 2012).

Las MA tienen dos componentes bien diferenciados: la “fase extrarradical” del hongo
en la que se incluyen el micelio externo, esporas y ocasionalmente células auxiliares, y
la “fase intrarradical” que incluye hifas intra e intercelulares, arbúsculos y, en algunas
especies, vesículas (Figura 4). La colonización de la raíz sólo ocurre en la epidermis y el
parénquima cortical, ya que el hongo nunca llega a penetrar en el cilindro vascular ni las
zonas meristemáticas (Barea et al., 2008).

26
Introducción

Figura 4. Esquema de las estructuras anatómicas de las MA. Abreviaturas: A, arbúsculo; AP,apresorio;
E, espora; H, hifa intercelular; M, micelio extraradical; O, ovillo; V, vesícula. (Tomado de Palenzuela
and Barea, 2002).

El proceso de formación de las MA tiene lugar tras una sucesión de interacciones entre
el hongo y la planta, que van a dar lugar a una integración morfológica y funcional de
ambos simbiontes (Gianinazzi-Pearson et al., 2004). Como resultado, la planta acepta la
colonización por parte del hongo sin mostrar una reacción de defensa persistente
(Dumas-Gaudot et al., 2000; Pozo et al., 2002). El establecimiento de la simbiosis es el
resultado de un continuo diálogo molecular y un esfuerzo mutuo para aceptar el
intercambio de señales de reconocimiento tanto de la planta como del hongo (Gollotte et
al., 2002; Vierheilig and Piché, 2002). La identificación y clonación de los genes de la
planta implicados en el programa simbiótico en MA (Gianinazzi-Pearson and
Brechenmacher, 2004) y el conocimiento de los programas de señales involucrados en
la formación y funcionamiento de la simbiosis (Harrison, 2005) son objeto de gran
interés en la actualidad.

27
Introducción

3.4. Significado de los hongos MA en el sistema suelo-planta

La mayoría de las plantas dependen de estar micorrizadas para establecerse y prosperar


adecuadamente. Ello es debido a que los hongos MA realizan importantes acciones en
los sistemas suelo-planta, como son las siguientes:

• Mejora del enraizamiento de las plantas


Mediante la producción de fitohormonas, vitaminas y otras sustancias fitoactivas por
parte de los hongos (Parniske, 2008; Faure et al., 2009).

• Incremento del suministro de nutrientes a las plantas


El micelio externo del hongo abarca una superficie mayor que las raíces de las plantas.
Es por ello que pueden captar nutrientes de zonas a las que la raíz no tiene acceso.
Además, nutrientes con poca movilidad y presentes en bajas concentraciones en el suelo
se absorben más eficientemente en plantas micorrizadas, acelerando los ciclos
biogeoquímicos de los nutrientes minerales, particularmente N y P (Ferrol and Pérez-
Tienda, 2009).

• Mejora de la estructura del suelo


Las hifas de los hongos MA están implicadas en la formación de agregados
hidroestables en el suelo, lo cual contribuye directamente en la mejora de la calidad del
suelo (Rillig and Mummey, 2006; Jeffries and Barea, 2012). La glomalina, una
glicoproteína producida por el micelio externo del hongo desempeña un importante
papel en el proceso de agregación (Rillig, 2004; Bedini et al., 2009).

• Protección a la planta frente a estreses abióticos


La simbiosis MA contribuye a incrementar la resistencia y tolerancia de las plantas a la
salinidad, sequía, estado de deficiencia o exceso de nutrientes, exceso de metales
pesados, degradación del suelo, etc (Barea et al., 2012).

• Protección a la planta frente a patógenos


Se ha descrito que las micorrizas reducen los síntomas de patógenos cuando se trata de
enfermedades que afectan al sistema radical (Cordier et al., 1996; Slezack et al., 2000).
Para que se manifieste esta protección, es imprescindible que la simbiosis esté
establecida antes de que se produzca el ataque del patógeno (Pozo et al., 2010)

• Favorecen la diversidad de las comunidades de plantas y la sucesión


vegetal
Cada planta muestra un nivel de compatibilidad mayor con determinados ecotipos de
hongos micorrícicos, por lo que la conservación de la diversidad de estos hongos

28
Introducción

beneficia la diversidad y sucesión de las plantas (van der Heijden et al., 1998; Hart and
Klironomos, 2002).

4. Efecto de la salinidad en las micorrizas arbusculares

La salinidad puede afectar negativamente a los hongos MA disminuyendo su capacidad


de colonización, la germinación de las esporas y el crecimiento de las hifas.
Varios estudios han demostrado que la salinidad puede disminuir la colonización de las
raíces por parte de los hongos MA (Duke et al., 1986; Giri et al., 2007; Sheng et al.,
2008), probablemente debido al efecto directo de la sal sobre el hongo (Juniper and
Abbott, 2006), lo que indica que la salinidad podría llegar a suprimir la formación de las
MA (Tian et al., 2004; Sheng et al., 2008). Los diferentes niveles de colonización MA
de las plantas bajo condiciones de salinidad se puede deber al diferente comportamiento
de cada especie de hongo MA, incluso en ecosistemas similares (Klironomos et al.,
1993) o a la influencia de diferentes condiciones ambientales (Carvalho et al., 2001). El
grado en el que la colonización MA se ve afectada por la salinidad es mayor al inicio
del proceso de colonización, cuando las esporas necesitan de hidratación para ser
activadas y producir el tubo de germinación. El exceso de sal en el suelo dificulta la
toma de agua y por tanto la hidratación de las esporas, siendo en este momento cuando
el exceso de sal retrasa la germinación (McMillen et al., 1998). El crecimiento de las
hifas también se ve reducido con el incremento de sal aunque se ha sugerido que el
crecimiento de las hifas puede considerarse más sensible al exceso de sal que la
germinación de las esporas, ya que la germinación se retrasa pero no necesariamente se
reduce mientras que el crecimiento de las hifas sí se ve reducido (Cantrell and
Linderman, 2001; Juniper and Abbott, 2006; Jahromi et al., 2008). La reducción en el
crecimiento de las hifas y retraso de la germinación de las esporas sugiere que si el
estrés salino persiste habría una reducción en el grado de colonización debido a que la
habilidad de infección de los propágulos se vería reducida.

Sin embargo, y en desacuerdo con lo anteriormente mencionado, hay estudios


que demuestran que la colonización MA no se reduce en presencia de sal (Levy et al.,
1983; Hartmond et al., 1987). Más recientemente, Yamato et al. (2008) y Wu et al.
(2010) demostraron que el grado de colonización de hongos MA no se vio reducido
incluso a altos niveles de salinidad. De hecho, las MA están ampliamente distribuidas
en ambientes salinos (Aliasgharzadeh et al., 2001; Carvalho et al., 2004; Yamato et al.,
2008; Wilde et al., 2009), lo que sugiere que las especies de hongos MA aisladas en
ambientes salinos deberían estar adaptadas a tales condiciones y esto explicaría el que
no disminuyera su capacidad colonizadora en presencia de sal en el medio.

29
Introducción

5. Simbiosis MA y tolerancia al estrés salino en plantas

Además de los mecanismos intrínsecos de adaptación a la salinidad desarrollados por


las plantas, en condiciones naturales crecen asociadas a las MA (Smith and Read, 2008).
En la mayoría de los casos estudiados, la asociación MA-planta confiere a la planta
hospedadora un incremento en la tolerancia ante determinados estreses abióticos (Barea
et al., 2012). Aunque algunos autores hayan señalado que el exceso de sales en el suelo
tiene un efecto negativo en las MA (Smith and Read, 1997; Juniper and Abbott, 2006),
muchos otros han demostrado que las MA incrementan la tolerancia de las plantas al
estrés salino (Al-Karaki et al., 2001; Garg and Manchanda, 2009; Hajiboland et al.,
2010).

La mejora en la tolerancia de las plantas colonizadas por MA al estrés salino se


puede deber en parte al resultado de una mayor eficiencia tanto en la toma de nutrientes
(Cantrell and Linderman, 2001; Asghari et al., 2005) como de agua (Augé, 2001; Aroca
et al., 2007); en la mejora del balance iónico (Zandavalli et al., 2004; Giri et al., 2007);
en una mayor producción de osmolitos (Sheng et al., 2011); en un aumento de la
fotosíntesis (Sheng et al., 2008) y en la protección de actividades enzimáticas (Giri and
Mukerji, 2004; Rabie and Almadini, 2005) entre otros mecanismos.

Todos estos estudios han sugerido varios mecanismos para explicar la mejora en la
tolerancia de las plantas al estrés salino:

- mejor eficiencia para la toma de agua y nutrientes, gracias a la red de


micelio que se extiende más allá de la zona de absorción directa de la
planta
- barrera selectiva durante la toma de nutrientes o transferencia a la planta
- mayor conductancia hidráulica de la raíz y ajuste osmótico
- incremento de la capacidad antioxidante
- mantenimiento de la relación K+/Na+ y menor acumulación de Na+ en las
hojas

Sin embargo los mecanismos que permiten a las plantas colonizadas por MA
tener mayor tolerancia a la salinidad están todavía lejos de ser comprendidos en su
totalidad. El mayor problema para resolver los mecanismos implicados en la mejora en
la tolerancia al la salinidad, es que la tolerancia salina es un mecanismo complejo y
codificado por familias de genes, lo que implica a varios mecanismos tanto fisiológicos
como bioquímicos y que además varían entre especies (Mian et al., 2011). Por lo tanto
los estudios del impacto de la colonización MA en la tolerancia de las plantas al estrés
salino se encuentran con este problema, entre otros. Además se debe prestar especial
atención a los aislados de MA en ambientes salinos, ya que deberían de estar fisiológica

30
Introducción

y genéticamente adaptados a dichas condiciones y por tanto mejorar la eficiencia de las


plantas sometidas a estrés salino (Ferrol et al., 2004). De hecho cada vez más estudios
demuestran la importancia de usar inóculo MA nativo de ambientes salinos tanto en la
mejora de la producción agrícola como en programas de revegetación en áreas afectadas
de salinidad (Marulanda et al., 2003; Moora et al., 2004; Cho et al., 2006; Alguacil et
al., 2011).

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6. Bibliografía

Al-Karaki GN, Hammad R, Rusan M. 2001. Response of two tomato cultivars


differing in salt tolerance to inoculation with mycorrhizal fungi under salt stress.
Mycorrhiza 11, 43-47.
Albaladejo J, Castillo V, Roldán A. 1996. Rehabilitation of degraded soils by water
erosion in semiarid environments. In: Rubio JL, Calvo A, eds. Soil degradation
and desertification in Mediterranean Environment. Logroño: Geoforma
Ediciones 265-278.
Alguacil MM, Torres MP, Torrecillas E, Díaz G, Roldán A. 2011. Plant type
differently promote the arbuscular mycorrhizal fungi biodiversity in the
rhizosphere after revegetation of a degraded, semiarid land. Soil Biology &
Biochemistry 43, 167-173.
Aliasgharzadeh N, Rastin NS, Towfighi H, Alizadeh A. 2001. Occurrence of
arbuscular mycorrhizal fungi in saline soils of the Tabriz Plain of Iran in relation
to some physical and chemical properties of soil. Mycorrhiza 11, 119-122.
Allen MF. 1991. The Ecology of Mycorrhizae. Cambridge: University Press.
Aroca R, Porcel R, Ruiz-Lozano JM. 2007. How does arbuscular mycorrhizal
symbiosis regulate root hydraulic properties and plasma membrane aquaporins
in Phaseolus vulgaris under drought, cold or salinity stresses? New Phytologist
173, 808-816.
Asensio Grima CM, Cantón Castilla Y, García Fernández I, Gil de Carrasco C,
Gómez Mercado F, de Haro Lozano S, Lozano Cantero FJ, del Moral
Torres F, Ortega Pérez R, Oyonarte Gutiérrez C, Pardo Martínez M,
Salvador Ramón M, Sánchez Garrido JA, Sánchez Gómez ST, Soriano
Rodríguez M. 2005. Almería. Factores formadores y suelos: Simón Torres, M.,
ed. Servicio de Publicaciones de la Universidad de Almería.
Asghari HR, Chittleborough DJ, Smith FA, Smith SE. 2005. Influence of arbuscular
mycorrhizal (AM) symbiosis on phosphorus leaching through soil cores. Plant
and Soil 275, 181-193.
Augé RM. 2001. Water relations, drought and vesicular-arbuscular mycorrhizal
symbiosis. Mycorrhiza 11, 3-42.
Bago B, Cano C. 2005. Breaking myths on arbuscular mycorrhizas in vitro biology. In
Vitro Culture of Mycorrhizas Vol 4. Soil Biology, pp. 111-138.
Barea JM. 1998. Biología de la rizosfera. Investigación y Ciencia (Scientific American)
256, 74-81.
Barea JM, Azcón-Aguilar C. 2012. Evolution, Biology and Ecological Effects of
arbuscular mycorrhizas. In: Gotsiridze-Columbus NS, ed. Symbiosis: Evolution,
Biology and Ecological Effects. USA: Nova Publishers.

32
Introducción

Barea JM, Ferrol N, Azcón-Aguilar C, Azcón R. 2008. Mycorrhizal symbioses. In:


White PJ, Hammond JP, eds. The Ecophysiology of Plant-Phosphorus
Interactions. Series: Plant Ecophysiology, Vol. 7 Dordrecht: Springer, 143-163.
Barea JM, Honrubia M. 1993. Micorrizas y revegetación. Ecosistemas 4, 46-47.
Barea JM, Jeffries P. 1995. Arbuscular mycorrhizas in sustainable soil plant systems.
In: Varma A, Hock B, eds. Mycorrhiza: Structure, Function, Molecular Biology
and Biotechnology. Heidelberg: Springer-Verlag 521-559.
Barea JM, Palenzuela J, Cornejo P, Sánchez I, Navarro C, Azcón R, Ferrol N,
Azcón-Aguilar C, Barea-Azcón JM, Travesí R, Moleón M, Ballesteros E,
Luzón JM, Tierno JM. 2006. Significado, diversidad e impacto de los hongos
de las micorrizas arbusculares en ambientes mediterráneos. In: Biodiversidad y
Conservación de Fauna y Flora en Ambientes Mediterráneos 2 Ed. Granada,
España: Sociedad Granatense de Historia Natural, 155-185.
Barea JM, Pozo MJ, López-Ráez JM, Aroca R, Ruíz-Lozano JM, Ferrol N, Azcón
R, Azcón-Aguilar C. 2012. Arbuscular Mycorrhizas and their significance in
promoting soil-plant systems sustainability against environmental stresses In:
Rodelas B, Gonzalez-Lopez J, eds. Beneficial Plant-Microbial Interactions:
Ecology and Applications USA: Science Publishers, (In press).
Bedini S, Pellegrino E, Avio L, Pellegrini S, Bazzoffi P, Argese E, Giovannetti M.
2009. Changes in soil aggregation and glomalin-related soil protein content as
affected by the arbuscular mycorrhizal fungal species Glomus mosseae and
Glomus intraradices. Soil Biology & Biochemistry 41, 1491-1496.
Blanca G, Morales C. 1991. Impactos sobre la flora y medidas de protección. In: Flora
del Parque Natural de la Sierra de Baza. Universidad de Granada: Servicio de
Publicaciones de la Universidad de Granada 349-362.
Boomsma CR, Vyn TJ. 2008. Maize drought tolerance: Potential improvements
through arbuscular mycorrhizal symbiosis? Field Crops Research 108, 14-31.
Brito I, de Carvalho M, Goss MJ. 2011. Summer survival of arbuscular mycorrhiza
extraradical mycelium and the potential for its management through tillage
options in Mediterranean cropping systems. Soil Use and Management 27, 350-
356.
Brundrett M. 2004. Diversity and classification of mycorrhizal associations. Biological
Reviews 79, 473-495.
Bucher M. 2007. Functional biology of plant phosphate uptake at root and mycorrhiza
interfaces. New Phytologist 173, 11-26.
Cantrell IC, Linderman RG. 2001. Preinoculation of lettuce and onion with VA
mycorrhizal fungi reduces deleterious effects of soil salinity. Plant and Soil 233,
269-281.
Carvalho LM, Caçador I, Martins-Louçao MA. 2001. Temporal and spatial variation
of arbuscular mycorrhizas in salt marsh plants of the Tagus estuary (Portugal).
Mycorrhiza 11, 303-309.

33
Introducción

Carvalho LM, Correia PM, Martins-Louçao MA. 2004. Arbuscular mycorrhizal


fungal propagules in a salt marsh. Mycorrhiza 14, 165-170.
Cho KH, Toler H, Lee J, Ownley B, Stutz JC, Moore JL, Auge RM. 2006.
Mycorrhizal symbiosis and response of sorghum plants to combined drought and
salinity stresses. Journal of Plant Physiology 163, 517-528.
Clark R, Zobel R, Zeto S. 1999. Effects of mycorrhizal fungus isolates on mineral
acquisition by Panicum virgatum in acidic soil. Mycorrhiza 9, 167-176.
Cordier C, Gianinazzi S, Gianinazzi-Pearson V. 1996. Colonisation patterns of root
tissues by Phytophthora nicotianae var parasitica related to reduced disease in
mycorrhizal tomato. Plant and Soil 185, 223-232.
Cornejo P, Rubio R, Castillo C, Azcón R, Borie F. 2008. Mycorrhizal effectiveness
on wheat nutrient acquisition in an acidic soil from southern chile as affected by
nitrogen sources. Journal of Plant Nutrition 31, 1555-1569.
Cornejo PE. 2006. Influencia de la cobertura vegetal sobre la diversidad y estructura de
las comunidades de hongos micorrícicos y sus efectos en la estabilización de
suelos degradados. Tesis Doctoral. Facultad de Ciencias. Universidad de
Granada, Granada.
del Val C, Barea JM, Azcón-Aguilar C. 1999. Diversity of arbuscular mycorrhizal
fungus populations in heavy-metal-contaminated soils. Applied and
Environmental Microbiology 65, 718-723.
Demidchik V, Maathuis FJM. 2007. Physiological roles of nonselective cation
channels in plants: from salt stress to signalling and development. New
Phytologist 175, 387-404.
Ding M, Hou P, Shen X, Wang M, Deng S, Sun J, Xiao F, Wang R, Zhou X, Lu C,
Zhang D, Zheng X, Hu Z, Chen S. 2010. Salt-induced expression of genes
related to Na+/K+ and ROS homeostasis in leaves of salt-resistant and salt-
sensitive poplar species. Plant Molecular Biology 73, 251-269.
Duke ER, Johnson CR, Koch KE. 1986. Accumulation of phosphorus, dry-matter and
betaine during NaCl stress of split-root citrus seedlings colonized with vesicular
arbuscular mycorrhizal fungi on zero, one or 2 halves. New Phytologist 104,
583-590.
Dumas-Gaudot E, Gollotte A, Cordier C, Gianinazzi S, Gianinazzi-Pearson V.
2000. Modulation of host defence systems. In: Kapulnik Y, Douds DD, Jr., eds.
Arbuscular Mycorrhizas: Physiology and Functions. Dordrecht, The
Netherlands: Kluwer Academic Publishers, 212-140.
Escribano P. 2002. Definition of Zonation units in Cabo de Gata-Níjar Natural Park.
Thesis Report GIRS-2002-045. Tesis Doctoral. Centre of Geo-Information and
Remote Sensing. Wageningen University, Wageningen.
Escribano P, Palacios-Orueta A, Oyonarte C. 2008. Cuantificación y distribución
espacial de los tipos de cubierta en los ecosistemas semiáridos con imágenes

34
Introducción

hiperespectrales, caso práctico en el Parque Natural Cabo de Gata-Níjar


(Almería) Ecosistemas 17.
Evelin H, Kapoor R, Giri B. 2009. Arbuscular mycorrhizal fungi in alleviation of salt
stress: a review. Annals of Botany 104, 1263-1280.
Faure D, Vereecke D, Leveau JHJ. 2009. Molecular communication in the
rhizosphere. Plant and Soil 321, 279-303.
Ferrol N, Calvente R, Cano C, Barea JM, Azcón-Aguilar C. 2004. Analysing
arbuscular mycorrhizal fungal diversity in shrub-associated resource islands
from a desertification-threatened semiarid Mediterranean ecosystem. Applied
Soil Ecology 25, 123-133.
Ferrol N, Pérez-Tienda J. 2009. Coordinated nutrient exchange in arbuscuar
mycorrhiza. In: Azcón-Aguilar C, Barea, J.M., Gianinazzi, S., Gianinazzi-
Pearson, V., eds. Mycorrhizas: Functional Processes and Ecological Impact:
Heidelberg: Springer-Verlag, 73-87.
Flowers TJ, Colmer TD. 2008. Salinity tolerance in halophytes. New Phytologist 179,
945-963.
Foyer CH, Noctor G. 2009. Redox regulation in photosynthetic organisms: signaling,
acclimation and practical implications. Antioxidants & Redox Signaling 11, 861-
905.
Francis DF, Thornes JB. 1990. Matorral, erosion and reclamation. In: (Eds)
Albaladejo J, Stocking MA, Díaz E, eds. In: Soil Degradation and
Rehabilitation in Mediterranean Environmental Conditions. Murcia, Spain:
CSIC, 87-115.
Frank AB. 1885. Ueber die auf Wurzelsymbiose beruhende Ernährung gewisser
Bäume durch Unteridische Pilze. Berichte der Deutschen Botanischen
Gesellschaft 3, 128-145.
Gallardo A, Covelo F, Morillas L, Delgado M. 2009. Ciclos de nutrientes y procesos
edáficos en los ecosistemas terrestres: especificidades del caso mediterráneo y
sus implicaciones para las relaciones suelo-planta. Ecosistemas 18 4-19.
Garg N, Manchanda G. 2009. Role of arbuscular mycorrhizae in the alleviation of
ionic, osmotic and oxidative stresses induced by salinity in Cajanus cajan (L.)
Millsp (pigeonpea). Journal of Agronomy and Crop Science 195, 110-123.
Geiger F. 1973. El Sureste español y los problemas de la aridez. Revista de geografia 7,
166-209.
Gerdemann JW, Trappe JM. 1974. The Endogonaceae in the Pacific Northwest.
Mycologia Memoir 5, 1-76.
Gianinazzi-Pearson V, Azcón-Aguilar C, Bécard G, Bonfante P, Ferrol N, Franken
P, Gollote A, Harrier L, Lanfranco L, van Tuinen D. 2004. Structural and
functional genomics of symbiotic arbuscular mycorrhizal fungi. In: Tkacz JS,
Lange L, eds. Advances in fungal biotechnology for industry, medicine and
agriculture. New York, Boston: Kluwer Academic/Plenum Publishers, 405-424.

35
Introducción

Gianinazzi-Pearson V, Brechenmacher L. 2004. Functional genomics of arbuscular


mycorrhiza: decoding the symbiotic cell programme. Canadian Journal of
Botany-Revue Canadienne de Botanique 82, 1228-1234.
Giri B, Kapoor R, Mukerji KG. 2007. Improved tolerance of Acacia nilotica to salt
stress by arbuscular mycorrhiza, Glomus fasciculatum may be partly related to
elevated K/Na ratios in root and shoot tissues. Microbial Ecology 54, 753-760.
Giri B, Mukerji K. 2004. Mycorrhizal inoculant alleviates salt stress in Sesbania
aegyptiaca and Sesbania grandiflora under field conditions: evidence for
reduced sodium and improved magnesium uptake. Mycorrhiza 14, 307-312.
Gollotte A, Brechenmacher L, Weidmann S, Franken P, Gianinazzi-Pearson V.
2002. Plant genes involved in arbuscular mycorrhiza formation and functioning.
In: Gianinazzi S, Schüepp H, Barea JM, Haselwandter K, eds. Mycorrhiza
Technology in Agriculture, from Genes to Bioproducts. Basel, Switzerland:
Birkhäuser Verlag, 87-102.
González-Guerrero M, Benabdellah K, Ferrol N, Azcón-Aguilar C. 2009.
Mechanisms underlying heavy metal tolerance in arbuscular mycorrhizas. In:
Azcón-Aguilar C, Barea JM, Gianinazzi S, Gianinazzi-Pearson V, eds.
Mycorrhizas Functional Processes and Ecological Impact. Berlin, Heidelberg:
Springer-Verlag, 107-122.
Greenway H, Munns R. 1980. Mechanisms of salt tolerance in nonhalophytes Annual
Review of Plant Physiology 31, 149-190.
Hajiboland R, Aliasgharzadeh N, Laiegh SF, Poschenrieder C. 2010. Colonization
with arbuscular mycorrhizal fungi improves salinity tolerance of tomato
(Solanum lycopersicum L.) plants. Plant and Soil 331, 313-327.
Harley JL, Smith SE. 1983. Mycorrhizal Symbiosis. New York: Academic Press.
Harrison MJ. 2005. Signaling in the arbuscular mycorrhizal symbiosis. Annual Review
of Microbiology 59, 19-42.
Hart M, Klironomos JN. 2002. Diversity of arbuscular mycorrhizal fungi and
ecosystem functioning. In: van der Heijden MGA, Sanders IR, eds. Mycorrhizal
Ecology. Berlin, Heidelberg: Springer, 225-242.
Hartmond U, Schaesberg NV, Graham JH, Syversten JP. 1987. Salinity and
flooding stress effects on mycorrhizal and non-mycorrhizal citrus rootstock
seedlings. Plant and Soil 104, 37-43.
Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ. 2000. Plant cellular and molecular
responses to high salinity. Annual Review of Plant Physiology and Plant
Molecular Biology 51, 463-499.
Hooker JE, Jaizme-Vega M, Atkinson D. 1994. Biocontrol of plant pathogens using
arbuscular mycorrhizal fungi. In: Gianinazzi S, Schüepp, H, eds. Impact of
Arbuscular Mycorrhizas on Sustainable Agriculture and Natural Ecosistems:
Birkhäuser-Verlag, Basel, Switzerland, 191-200.

36
Introducción

IPCC. 2007. Intergovernmental Panel on Climate Change, Synthesis Report,


Contribution of Working Groups I, II and III to the Fourth Assessment Report of
the Intergovernmental Panel on Climate Change IPCC, Geneva.
Jahromi F, Aroca R, Porcel R, Ruiz-Lozano JM. 2008. Influence of salinity on the In
vitro development of Glomus intraradices and on the In vivo physiological and
molecular responses of mycorrhizal lettuce plants. Microbial Ecology 55, 45-53.
Jeffries P, Barea JM. 2012. Arbuscular Mycorrhiza - a key component of sustainable
plant-soil ecosystems. In: Hock B, ed. The Mycota, vol IX. Fungal Associations,
2nd edition. Berlin, Heidelberg: Springer-Verlag, 95-113.
Juniper S, Abbott L. 2006. Soil salinity delays germination and limits growth of
hyphae from propagules of arbuscular mycorrhizal fungi. Mycorrhiza 16, 371-
379.
Klironomos JN, Moutoglis P, Kendrick B, Widden P. 1993. A comparison of spatial
heterogeneity of vesicular-arbuscular mycorrhizal fungi in two maple forest
soils. Canadian Journal of Botany 71, 1472-1480.
Krüger M, Krüger C, Walker C, Stockinger H, Schussler A. 2012. Phylogenetic
reference data for systematics and phylotaxonomy of arbuscular mycorrhizal
fungi from phylum to species level. New Phytologist 193, 970-984.
Levy Y, Dodd J, Krikun J. 1983. Effect of irrigation water salinity and rootstock on
the vertical distribution of vesicular-arbuscular mycorrhiza in citrus roots. New
Phytologist 95, 397-403.
López-Bermúdez F, Albaladejo J. 1990. Factores ambientales de la degradación del
suelo en el area mediterranea. In: Albaladejo J, Stocking, M.A., Díaz, E., eds.
Degradation and rehabilitation of soil in mediterranean environmental
conditions. Murcia, Spain: CSIC, 15-45.
López-González GA. 2001. Los árboles y arbustos de la península ibérica y baleares
(especies silvestres y las principales cultivadas). España: Edic. Mundi-Prensa.
Maathuis FJM, Amtmann A. 1999. K+ nutrition and Na+ toxicity: the basis of cellular
K+/Na+ ratios. Annals of Botany 84, 123-133.
Mahajan S, Tuteja N. 2005. Cold, salinity and drought stresses: an overview. Archives
of Biochemistry and Biophysics 444, 139-158.
Marschner H. 1995. Mineral nutrition of higher plants. 2nd edition London, UK:
Academic Press.
Martínez LB, Pugnaire FI. 2009. Interacciones entre las comunidades de hongos
formadores de micorrizas arbusculares y de plantas. Algunos ejemplos en los
ecosistemas semiáridos. Ecosistemas 18, 44-54.
Marulanda A. 2006. Estudio de los mecanismos implicados en la resistencia de las
plantas a estreses osmóticos inducidos por microorganismos autóctonos
promotores del crecimiento vegetal (hongos micorrízicos arbusculares y
bacterias). Tesis Doctoral. Facultad de Ciencias. Universidad de Granada,
Granada.

37
Introducción

Marulanda A, Azcón R, Ruíz-Lozano JM. 2003. Contribution of six arbuscular


mycorrhizal fungal isolates to water uptake by Lactuca sativa plants under
drought stress. Physiologia Plantarum 119, 526-533.
McMillen B, Juniper S, Abbott LK. 1998. Inhibition of hyphal growth of a vesicular-
arbuscular mycorrhizal fungus in soil containing sodium chloride limits the
spread of infection from spores. Soil Biology and Biochemistry 30, 1639-1646.
Mian AA, Senadheera P, Maathuis FJM. 2011. Improving crop salt tolerance: anion
and cation transporters as genetic engineering targets. Plant Stress 5, 64-72.
Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R. 2010. Reactive oxygen species
homeostasis and signalling during drought and salinity stresses. Plant Cell and
Environment 33, 453-467.
Mittler R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends in Plant
Science 7, 405-410.
Mittler R, Vanderauwera S, Gollery M, van Breusegem F. 2004. Reactive oxygen
gene network of plants. Trends in Plant Science 9, 490-498.
Moller IM, Jensen PE, Hansson A. 2007. Oxidative modifications to cellular
components in plants. Annual Review of Plant Biology 58, 459-481.
Montalvo J. 1992. Interpretación ecológica de la erosión y desertificación. Ecosistemas
3, 17.
Moora M, Öpik M, Sen R, Zobel M. 2004. Native arbuscular mycorrhizal fungal
communities differentially influence the seedling performance of rare and
common Pulsatilla species. Functional Ecology 18, 554-562.
Munns R. 2002. Comparative physiology of salt and water stress. Plant Cell &
Environment 25, 239-250.
Munns R. 2005. Genes and salt tolerance: bringing them together. New Phytologist
167, 645-663.
Munns R, Tester M. 2008. Mechanisms of salinity tolerance. Annual Review of Plant
Biology 59, 651-681.
Nandwal AS, Kukreja S, Kumar N, Sharma PK, Jain M, Mann A, Singh S. 2007.
Plant water status, ethylene evolution, N2 fixing efficiency, antioxidant activity
and lipid peroxidation in Cicer arietimum L. nodules as affected by short term
salinization and desalinization Journal of Plant Physiology 164, 1161-1169.
Oehl F, Sieverding E, Palenzuela J, Ineichen K, Silva GA. 2011. Advances in
Glomeromycota taxonomy and classification. IMA Fungus 2, 191-199.
Oliveira RS, Vosátka M, Dodd JC, Castro PML. 2005. Studies on the diversity of
arbuscular mycorrhizal fungi and the efficacy of two native isolates in a highly
alkaline anthropogenic sediment. Mycorrhiza 16, 23-31.
Palenzuela J, Barea JM. 2002. Técnicas empleadas en el estudio de las micorrizas de
plantas endémicas del tomillar dolomitícola del Parque Natural de la Sierra de
Baza. Acta Granatense 2, 125-138.

38
Introducción

Parniske M. 2008. Arbuscular mycorrhiza: the mother of plant root endosymbioses.


Nature Reviews Microbiology 6, 763-775.
Pitman M, Läuchli A. 2002. Global impact of salinity and agricultural ecosystems. In:
Läuchli A, Lüttge U, eds. Salinity: environment-plants-molecules. Netherlands:
Kluwer Academic Publishers, 3-20.
Pozo MJ, Cordier C, Dumas-Gaudot E, Gianinazzi S, Barea JM, Azc¢n-Aguilar C.
2002. Localized versus systemic effect of arbuscular mycorrhizal fungi on
defence responses to Phytophthora infection in tomato plants. Journal of
Experimental Botany 53, 525-534.
Pozo MJ, Jung SC, López-Ráez JA, Azcón-Aguilar C. 2010. Impact of arbuscular
mycorrhizal symbiosis on plant response to biotec stress: The role of plant
defence mechanisms. In: Koltai H, Kapulnik Y, eds. Arbuscular Mycorrhizas:
Physiology and Function. Springer, Netherlands., 193-207.
Pozo MJ, Verhage A, García-Andrade J, García JM, Azcón-Aguilar C. 2009.
Priming plant defence against pathogens by arbuscular mycorrhizal fungi. In:
Azcón-Aguilar C, Barea JM, Gianinazzi S, Gianinazzi-Pearson V, eds.
Mycorrhizas Functional Processes and Ecological Impact. Berlin, Heidelberg:
Springer-Verlag, 123-135.
Rabie GH, Almadini AM. 2005. Role of bioinoculants in development of salt-
tolerance of Vicia faba plants under salinity stress. African Journal of
Biotechnology 4, 210-222.
Redecker D, Kodner R, Graham LE. 2000. Glomalean fungi from the Ordovician.
Science 289, 1920-1921.
Requena N, Jeffries P, Barea JM. 1996. Assessment of natural mycorrhizal potential
in a desertified semiarid ecosystem. Applied and Environmental Microbiology
62, 842-847.
Requena N, Pérez-Solis E, Azcón-Aguilar C, Jeffries P, Barea JM. 2001.
Management of indigenous plant-microbe symbioses aids restoration of
desertified ecosystems. Applied and Environmental Microbiology 67, 495-498.
Rillig MC. 2004. Arbuscular mycorrhizae, glomalin, and soil aggregation. Canadian
Journal of Soil Science 84, 355-363.
Rillig MC, Mummey DL. 2006. Mycorrhizas and soil structure. New Phytologist 171,
41-53.
Rivas-Martínez S. 1981. Les étages bioclimatiques de la végétation de la Péninsule
Ibérique. Anales del Jardín Botánico de Madrid. 37, 251-268.
Rodríguez P, Torrecillas A, Morales MA, Ortuño MF, Sánchez-Blanco MJ. 2005.
Effects of NaCl salinity and water stress on growth and leaf water relations of
Asteriscus maritimus plants. Environmental and Experimental Botany 53, 113-
123.
Ruiz-Lozano JM, Aroca R. 2010. Host response to osmotic stresses: stomatal
behaviour and water use efficiency of arbuscular mycorrhizal plants. In: Koltai

39
Introducción

H, Kapulnik Y, eds. Arbuscular Mycorrhizas: Physiology and Function, 2nd Ed.


Dordrecht, The Netherlands: Springer Science, Business Media B.V. , 239-256.
Ruiz-Lozano JM, Porcel R, Azcón R, Aroca R. 2012. Regulation by arbuscular
mycorrhizae of the integrated physiological response to salinity in plants: new
challenges in physiological and molecular studies. Journal of Experimental
Botany 63, 4033-4044.
Sánchez-Castro I. 2009. Análisis de la estructura y diversidad de las comunidades de
hongos formadores de micorrizas arbusculares asociados a plantas de especial
interés ecológico en ambientes mediterráneos. Tesis Doctoral. Facultad de
Ciencias. Universidad de Granada, Granada.
Sánchez-Castro I, Ferrol N, Barea JM. 2012. Analyzing the community composition
of arbuscular mycorrhizal fungi colonizing the roots of representative shrubland
species in a Mediterranean ecosystems Journal of Arid Environments 80, 1-9.
Scheibe R, Beck E. 2011. Drought, desiccation, and oxidative stress. In: Lüttge U,
Beck E, Bartels D, eds. Plant Desiccation Tolerance, Ecological Studies. Berlin:
Springer-Verlag, 209-231.
Schüβler A, Schwarzott D, Walker C. 2001. A new fungal phylum, the
Glomeromycota, phylogeny and evolution. Mycological Research 105, 1413-
1421.
Serrano A. 2009. Efecto de diferentes factores: fertilización, salinidad y procesado,
sobre parámetros objetivos de calidad en pimiento.Tesis Doctoral. Departamento
de Tecnología de la Alimentación y Nutrición. Univesidad Católica San
Antonio, Murcia.
Shabala S, Cuin TA. 2008. Potassium transport and plant salt tolerance. Physiologia
Plantarum 133, 651-669.
Sheng M, Tang M, Chen H, Yang B, Zhang F, Huang Y. 2008. Influence of
arbuscular mycorrhizae on photosynthesis and water status of maize plants
under salt stress. Mycorrhiza 18, 287-296.
Sheng M, Tang M, Zhang FF, Huang YH. 2011. Influence of arbuscular mycorrhiza
on organic solutes in maize leaves under salt stress. Mycorrhiza 21, 423-430.
Slezack S, Dumas-Gaudot E, Paynot M, Gianinazzi S. 2000. Is a fully established
arbuscular mycorrhizal symbiosis required for bioprotection of Pisum sativum
roots against Aphanomyces euteiches? Molecular Plant-Microbe Interactions
13, 238-241.
Smith SE, Read DJ. 1997. Mycorrhizal symbiosis, 2nd ed. London, UK: Academic
Press.
Smith SE, Read DJ. 2008. Mycorrhizal Symbiosis, 3rd Ed. New York: Elsevier,
Academic Press.
Teakle NL, Tyerman SD. 2010. Mechanisms of Cl- transport contributing to salt
tolerance. Plant, Cell & Environment 33, 566-589.

40
Introducción

Tester M, Davenport R. 2003. Na+ tolerance and Na+ transport in higher plants.
Annals of Botany 91, 503-527.
Therond P, Bonnefont-Rousselot D, Davit-Spraul A, Conti M, Legrand A. 2000.
Biomarkers of oxidative stress: an analitical approach. Current Opinion in
Clinical Nutrition and Metabolic Care 3, 373-384.
Thompson JD, Lavergne S, Affre L, Gaudeul M, Debussche M. 2005. Ecological
differentiation of Mediterranean endemic plants. Taxon 54, 967-976.
Tian CY, Feng G, Li XL, Zhang FS. 2004. Different effects of arbuscular mycorrhizal
fungal isolates from saline or non-saline soil on salinity tolerance of plants.
Applied Soil Ecology 26, 143-148.
Tuteja N. 2007. Mechanisms of high salinity tolerance in plants. Methods in
Enzymology 428, 419-438.
van der Heijden MGA, Bardgett RD, van Straalen NM. 2008. The unseen majority:
soil microbes as drivers of plant diversity and productivity in terrestrial
ecosystems. Ecology Letters 11, 296-310.
van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R,
Boller T, Wiemken A, Sanders IR. 1998. Mycorrhizal fungal diversity
determines plant biodiversity, ecosystem variability and productivity. Nature
396, 69-72.
Vierheilig H, Piché Y. 2002. Signalling in arbuscular mycorrhiza: Facts and
hypotheses. Flavonoids in Cell Function 505, 23-39.
Wheeler JH, Kostbade JT. 1990. World Regional Geography: Saunders College
Publishing.
White PJ, Broadley MR. 2001. Chloride in soils and its uptake and movement within
the plant: a review. Annals of Botany 88, 967-988.
Wilde P, Manal A, Stodden M, Sieverding E, Hildebrandt U, Bothe H. 2009.
Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt
marshes. Environmental Microbiology 11, 1548-1561.
Wright SF, Upadhyaya A. 1998. A survey of soils for aggregate stability and
glomalin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi.
Plant and Soil 198, 97-107.
Wu QS, Zou YN, He XH. 2010. Contributions of arbuscular mycorrhizal fungi to
growth, photosynthesis, root morphology and ionic balance of citrus seedlings
under salt stress. Acta Physiologiae Plantarum 32, 297-304.
Xu G, Magen H, Tarchitzky J, Kafkafi U. 2000. Advances in chloride nutrition.
Advances in Agronomy 68, 96-150.
Yaalon DH. 1997. Soils in the Mediterranean region: What makes them different?
Catena 28, 157-169.
Yamato M, Ikeda S, Iwase K. 2008. Community of arbuscular mycorrhizal fungi in a
coastal vegetation on Okinawa island and effect of the isolated fungi on growth
of sorghum under salt-treated conditions. Mycorrhiza 18, 241-249.

41
Introducción

Zandavalli RB, Dillenburg LR, de Souza PVD. 2004. Growth responses of Araucaria
angustifolia (Araucariaceae) to inoculation with the mycorrhizal fungus Glomus
clarum. Applied Soil Ecology 25, 245-255.
Zhu JK. 2003. Regulation of ion homeostasis under salt stress. Current Opinion in
Plant Biology 6, 441-445.

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CAPÍTULO 1
CHAPTER 1

Diversity of arbuscular mycorrhizal fungi in the rhizosphere of


Asteriscus maritimus (L), a representative plant species in arid and
saline Mediterranean ecosystems

Submitted to Applied Soil Ecology by Estrada B, Beltrán-Hermoso M,


Palenzuela J, Iwase K, Ruiz-Lozano J.M, Barea J.M and Oehl F

33
34
Capítulo 1

Diversidad de hongos micorrícico arbusculares en la rizosfera de


Asteriscus maritimus (L), una planta representativa de ecosistemas
Mediterráneos áridos y salinos

Resumen

El uso de los hongos MA adaptados a la salinidad puede ser un elemento crucial en la


recuperación de áreas salinas, ya sea en entornos naturales o en terrenos agrícolas que se
han salinizado debido a un uso no adecuado del suelo o bajo un escenario de cambio
climático. Por lo tanto, la búsqueda de hongos MA adaptados a la salinidad es
fundamental para explorar esas posibilidades. A pesar de su importancia, existe poca
información sobre la distribución y abundancia de las diferentes asociaciones de
micorrizas en ambientes salinos en zonas europeas del Mediterráneo. En el presente
estudio se investiga la comunidad de hongos MA en la rizosfera de una especie de
planta representativa adaptada a zonas salinas mediterráneas, Asteriscus maritimus (L.).
Se tomaron muestras de la rizosfera de veinte plantas de A. maritimus en dos zonas
diferentes del Parque Natural de Cabo de Gata: una duna salina y una marisma. Un total
de 30 morfotipos de esporas pertenecientes a tres clases, cinco órdenes, nueve familias y
13 géneros fueron encontrados en la duna y en la marisma. En la rizosfera de la
marisma la densidad de esporas fue seis veces mayor que en la duna, a pesar de que la
diversidad fue bastante similar en ambos sitios. Una nueva especie de hongo MA se ha
descrito en la duna y publicado recientemente, y cuatro especies no pudieron ser
identificadas de forma inequívoca, lo que sugiere posibles nuevas especies que aún
necesitan un análisis más detallado antes de su publicación. Estos resultados deben ser
tomados en cuenta en el diseño de inóculos eficaces en programas de revegetación.

Palabras clave: biodiversidad, micorriza arbuscular, Mediterráneo, salinidad, nativo,


inóculo

45
46
Chapter 1

Diversity of arbuscular mycorrhizal fungi in the rhizosphere of


Asteriscus maritimus (L), a representative plant species in arid and
saline Mediterranean ecosystems

Beatriz Estradaa, Maria Beltrán-Hermosob, Javier Palenzuelaa, Koji Iwasec, Juan Manuel
Ruiz-Lozanoa, José-Miguel Bareaa* and Fritz Oehld*

a
Departamento de Microbiología del Suelo y Sistemas Simbióticos. Estación Experimental del
Zaidín (CSIC). Profesor Albareda nº 1, 18008 Granada, Spain.
b
Departamento de Ingeniería Civil, Edificio Politécnico, Universidad de Granada, Campus de
Fuentenueva, 18071, Spain.
c
Department of Natural and Environmental Science, Teikyo University of Science,
2525 Yatsusawa, Uenohara 409-0193, Japan.
d
Agroscope Reckenhoz-Tänikon Research Station (ART), Ecological Farming Systems,
Reckenholzstrasse 191, CH-8046, Zürich, Switzerland.

* Corresponding authors: Prof. José-Miguel Barea


e-mail: josemiguel.barea@eez.csic.es
Tel: + 34 958 181600
Fax: + 34 958 129600
Dr. Fritz Oehl
e-mail: Fritz.Oehl@art.admin.ch
Tel: + 41 (0)44 377 7321
Fax: + 41 (0)44 377 7201

47
Chapter 1

Abstract

The use of AM fungi adapted to salinity could be a critical issue for success in
recovering saline areas either in natural environments or in agricultural lands that
became salinized due to a non appropriate land use or under a climate change scenario.
Therefore looking for salinity-adapted AM fungi is fundamental to explore these
possibilities. Despite its important role, there is little information on the distribution and
abundance of the different mycorrhizal associations in saline environments in European
Mediterranean areas. In the present study the community of AM fungi is investigated in
the rhizosphere of a representative plant species adapted to saline Mediterranean areas,
Asteriscus maritimus (L.). Samples of the rhizosphere of twenty A. maritimus plants
were taken in two different areas of the Cabo de Gata Natural Park: a saline dune and a
salt marsh. A total of 30 spore morphotypes belonging to three classes, five orders, nine
families and 13 genera were found in the dune and salt marsh. In the rhizosphere of the
salt marsh spore densities were six times higher than in the dunes, although the diversity
was quite similar in both systems. One new AM fungal species has been described in
the dune and recently published, and four species could not been unequivocally
identified, suggesting possible new species that still need some further analysis before
publication. These results should be taken into account when designing effective
inocula for revegetation programmes.

Key words: biodiversity, arbuscular mycorrhiza, Mediterranean, salinity, native,


inoculum.

1. Introduction

Seasonal aridity characterises Mediterranean environments. Impoverished soils, extreme


temperatures, irregular rainfall, soil misuse and overgrazing for cattle-raising are several
of the factors which contribute to the eroded landscapes (Vallejo et al. 1999). This
multiple stress situation is a constraint for plant development because damage to soil
and plants in arid and semiarid areas is not easily repaired due to the fragility of these
ecosystems (Pascual et al. 2000). The latter is promoting desertification of great areas in
Southeast Spain which leads to increases in salinity (Francis and Thornes 1990; López-
Sánchez and Honrubia 1992; Albaladejo et al. 1996). Disturbance of the vegetation
cover is the first visible indication of a desertification process and it is known to occur
concomitantly with generalised damages of physical-chemical and biological soil
properties (Requena et al. 2001). Moreover, salinity is recognized as a severe global
ecological problem being one of the most influential abiotic factors limiting plant
growth and yield (Porcel et al., 2012).

48
Chapter 1

In these areas, where conditions are unfavourable for plant growth, arbuscular
mycorrhizal (AM) fungi play an essential role (Trappe 1981). AM fungi are ubiquitous
soil inhabitants belonging to the phylum Glomeromycota which establish mutualistic
symbiotic associations with most land plants (Smith and Read 2008). The symbiosis
between plant and AM fungi is one of the plant strategies for growing under a variety of
stress conditions (Entry et al. 2002). The ecological impact of AM fungi is particularly
relevant for arid and semi-arid ecosystems where they would enable greater plant
tolerance of environmental stresses characteristic of these ecosystems (Requena et al.
1996; Allen 2007).
It is well documented that AM fungi improve plant growth and health: they facilitate
nutrient uptake (van der Heijden et al. 2006), confer drought and salt tolerance (Ruíz-
Lozano and Azcón 2000; Cantrell and Linderman 2001; Marulanda et al. 2009), protect
plants against pathogens (Slezack et al. 2000; Pozo et al. 2009), have positive impacts
on soil aggregate stability and water infiltration (Rillig and Mummey 2006), and
prevent soil erosion (O'Dea 2007). Because of the key ecological functions played in
soil and plants by AM symbiosis, a diverse community of AM fungi is necessary for the
development and maintenance of plant diversity (Jeffries and Barea 2001), contributing
to plant community productivity in different ecosystems (van der Heijden et al. 1998).
Therefore, as AM fungi play important roles in the vigour of plant communities and the
restoration of disturbed ecosystems (Renker et al. 2004), assessment of the native
species composition is an important issue in several contexts and the basis to produce
AM inoculum for selected plant species to be used in the revegetation processes (Ferrol
et al. 2004). Moreover, the use of AM fungi adapted to salinity could be a critical issue
for success in recovering saline areas either in natural environments or in agricultural
lands that became salinized due to inappropriate land use. Therefore looking for
salinity-adapted AM fungi is fundamental to explore these possibilities (Porcel et al.,
2012; Estrada et al., 2012). Despite its important role, there is little information on the
distribution and abundance of the different mycorrhizal associations in saline
environments in European Mediterranean areas.

The aim of the present work was to analyse the diversity of AM fungal species in
sensitive ecosystems affected by desertification and salinity in Southeast Spain,
selecting two ecosystems: salt marshes and dunes. Cabo de Gata Natural Park is the
most arid ecosystem in Europe (Geiger 1973) and was selected as the target area. AM
fungi were screened in the rhizosphere of Asteriscus maritimus (L.), an halophyte
member of the Asteraceae family, highly mycotrophic (Schaede 1962), and native of
lands surrounding the Mediterranean Sea, especially Spain (Lendínez et al. 2011).
Moreover, as the plant spreads its stems over the soil they protect the soil from erosion
(Alcaraz et al. 1997). It is found both in salt marshes and dunes. However, differences
in the AM fungal communities associated with this plant can be assumed dependent on
the ecosystems. For this purpose AM fungi were studied either directly in samples from

49
Chapter 1

the target soil or from one year bait plant cultures established to increase AM fungal
spore population, to reveal their maximum complement possible.
To identify the different AM fungal species in natural saline sites, morphological and
developmental criteria were applied. The morphological identification of AMF can
accurately be done with spores (Oehl et al. 2011), while the fungal structures within the
roots (intraradical hyphae, arbuscules, vesicles,) may vary only within the family or
order but not at the species level (Dodd et al. 2000). The molecular characterization of
AMF in roots is faced with the problem that only about 5% of the total DNA of a root
belongs to the fungi (Toth et al. 1991). Our primary purposes were to understand the
species composition of AM fungi in the rhizosphere of A. maritimus, and to elucidate
the distribution patterns of AM fungi communities related to habitats.

2. Material and Methods

2.1. Site description

The study was carried out in a representative saline Mediterranean ecosystem in the
Natural Park Cabo de Gata in Almería (Andalucía, Spain). The climate is dry and hot,
with an average annual temperature of 18.5ºC, irregular rainfall occurring mostly in
autumn with a mean annual rainfall less than 200mm and an annual solar hours of
2.960. Two habitats were selected: a natural sand dune (36º44´41´´N 02º07´26´´W) and
a salt marsh (36º45´24´´N 02º13´17´´W).

2.2. Plant and soil sampling

Most of the halophytes in saline sites belong to the Chenopodiaceae, Juncaceae,


Cyperaceae or Brassicaceae families which are non-or weakly mycorrhizal. For the
present study the halophyte Asteriscus maritimus, belonging to the Asteraceae family
was selected as target plant which is known to be colonized by AMF since long time
(Mason 1928).
Soil samples were taken from the rhizosphere of twenty A. maritimus (L.) plants at both
sites in February 2010. Plants were randomly collected with their intact root systems up
to 40 cm soil depth, ten of them growing in the natural sand dune system and the other
ten plants in the salt marsh. Root fragments in these samples were rinsed cleansed with
tap water and then cleared in 10% KOH and stained with 0.05% trypan blue to confirm
AM colonization (Phillips and Hayman 1970). The samples were further used to analyse
spore populations and selected chemical soil parameters (pH, soil organic carbon, total
nitrogen, soil electrical conductivity).

50
Chapter 1

2.3. AM fungal bait and pure cultures

AM fungal bait cultures were also established and maintained as described in


Palenzuela et al. (2008) and Palenzuela et al. (2011) by transplanting each A. maritimus
plants with their surrounding rhizosphere soils into 1 L pots and transferring them to the
greenhouse at EEZ in Granada immediately after sampling. The pots were irrigated
three times per week and fertilized every four weeks with Long-Aston nutrient solution
(Hewitt 1952). The trap cultures were used for propagation of indigenous AM fungal
communities to be checked after one year. With the main species present in these
habitats pure cultures were established at the EEZ collection for future experiments and
rehabilitation programs of saline areas using the benefits of native AM fungi.

2.4. AMF spore isolation and identification

For the analyses of AM fungal spores, 50 g of each rhizospheric soil were taken in
March 2010, and 25 g in March 2011 from each trap cultures. AM fungal spores, both
from the field collected soil and from the trap cultures, were isolated, identified and
counted separately for each location. They were separated from the soil samples by a
wet sieving and decanting method, followed by sucrose centrifugation process
(Sieverding 1991). After centrifugation, the supernatant was poured through a 50 μm
mesh and quickly rinsed with tap water. Turgid spores (suggesting viability) were
counted under a dissecting microscope using up to 90-fold magnification. For
identification of AMF species, spores (about 50-70% of the total numbers) were picked
under the dissecting microscope and mounted in polyvinyl alcohol-lactic acid-glycerine
(PVLG) (Koske and Tessier 1983) or PVLG mixed 1:1 (v/v) with Melzer's reagent
(Brundrett et al. 1994) for permanent slides. The spores were then examined using a
compound microscope at up to 400-fold magnification. The AMF species identification
was based on current species descriptions and identification manuals (e.g. Schenck and
Pérez 1990); International Culture Collection of (Vesicular) Arbuscular Mycorrhizal
Fungi (http://invam.caf.wvu.edu/fungi/taxonomy/speciesID.htm); Department of Plant
Pathology University of Agriculture in Szczecin, Poland
(http://www.agro.ar.szczecin.pl/~jblaszkowski/)), and on own type specimen analyses
of almost all AMF species described. In this study, an AMF “species” is either a clearly
identified species based on its spore morphology, or a species as yet unknown to us. We
follow the glomeromycotean classification of Oehl et al. (2011) recently published in
the new journal of the International Mycological Association on request. Photographs
were taken with a Leica DFC 290 digital camera on a Leitz Laborlux S compound
microscope using Leica Application Suite Version V 2.5.0 R1 software.

51
Chapter 1

2.5. Species richness and specific density

The species richness is defined as the total number of species found in a community.
The specific density (Di) is the proportion of individuals of one particular species in the
sample (i) relative to the total number of individuals (N).
Di = ni / N
In the present work species richness and specific density have been evaluated at the
dune and salt marsh.

3. Results

3.1. Root colonization by AMF

All surveyed samples were colonized by AMF and formed typical arbuscular
mycorrhizal structures. Intra- and intercellular hyphae, vesicles and arbuscules were
abundant in the root tissues.

3.2. Soil analysis

At the dune site, the soil was alkaline with pH 8.2, organic carbon 15.3 g kg-1, total
nitrogen 1.9 g kg-1, available P 27.0 mg kg-1, and soil electrical conductivity 0.5 dS m-1.
At the salt marsh, the soil was alkaline with pH 8.7, organic carbon 2.6 g kg-1, total
nitrogen 0.3 g kg-1, available P 47.0 mg kg-1 and soil electrical conductivity 3.95 dS m-1.

3.3. AMF spore densities at different ecosystems

The AMF spore densities differed among the sites. Salt marsh had regularly higher
spore densities than the dune, both at the rhizosphere soil and in the bait cultures. In
March 2010, the spore densities were 1.2 and 7.2 g-1 soil at the dune and the salt marsh,
respectively. After one year in the trap cultures, the spores densities were more than
doubled, with 2.8 and 17.8 spores at the dune and the salt marsh. The relation between
dune and salt marsh remained the same, being the spore density around six times higher
at the salt marsh than at the dune.

3.4. AMF species richness

In total 30 AMF species could be distinguished from the field samples across all sites
and both years of survey. They belong to all three current classes, to all five current
orders, to nine families and thirteen genera currently known in the Glomeromycota
(Table 1). The majority of the isolated species (16) were of the order Glomerales (3

52
Chapter 1

Funneliformis species, 9 Glomus species and one Septoglomus species of the


Glomeraceae and two species of Claroideoglomus and one Entrophospora species of
the Entrophosporaceae). One of them, Glomus rubiforme, had formerly been assigned to
Sclerocystis. Of the Diversisporales, four Diversispora, two Pacispora and one
Acaulospora were detected. Of the Gigasporales, two Racocetra and one Scutellospora
were found. Finally, two Archaeospora and one Ambispora species of the Ambisporales
and one Paraglomus species of the Paraglomerales were detected. Four species (Glomus
sp. BEV1, Gl. sp. BEV2, Entrophospora sp. BEV3 and Diversispora sp. BEV4) could
not be unequivocally identified as a known species and might represent new species.

3.5. AMF species richness in sand dune

In the field samples of March 2010, 16 AMF species belonging to 9 genera were found
in the sand dune. After one year of bait culturing, 15 AMF species reproduced spores in
the cultures belonging to 10 genera. From field and bait cultures, a total of 23 AMF
species were detected belonging to 11 AMF genera were identified. Of these species, 7
were found recovered only from the field samples and 8 only from the bait cultures,
while 9 species were detected both in the field samples and the bait cultures (Table 1).
In 2010, the species detected (in order of decreasing specific density), were: Se.
constrictum, Diversispora sp. BEV4, Cl. claroideum, Fu. coronatus, Gl. badium, Gl.
intraradices, Gl. macrocarpum, Gl. sp. BEV2, Sc. calospora, Gl. rubiforme, Ra.
persica, Ac. scrobiculata. After one year of bait culturing, the species recovered from
the sand dune (in order of decreasing specific density, see Figure 1) were: Se.
constrictum, Fu. coronatus, Cl. claroideum, Pa. dominikii, Gl. intraradices, Cl.
etunicatum, Di. clara, Di. versiformis, Fu. mosseae, Gl. microaggregatum, Sc.
calospora, Di. aurantia, Par. occultum, Ar. trappei, Pa. franciscana, Ac. scrobiculata.
In the field and in the bait cultures, Se. constrictum was the most abundant species.

3.6. AMF species richness in salt marsh

In the salt marsh system, a total of 24 AMF species, also belonging to 11 AMF genera,
were detected (Table 1). In the field samples from the salt marsh, 20 morphospecies
were identified in 2010 belonging to 10 AMF genera, and after one year of bait
culturing, 12 AMF species had reproduced spores belonging to 7 genera. Of these
species, 11 were found identified only from the field samples and 4 only from the bait
cultures, while 9 species were recovered from both the field samples and the bait
cultures.
In 2010, the species detected (in order of decreasing specific density, see Figure 1),
were: Se. constrictum, Diversispora sp. BEV4, Gl. sp. BEV2, Entrophospora sp. BEV3,
Gl. intraradices, Cl. claroideum, Fu. coronatus, Sc. calospora, Gl. macrocarpum, Fu.
mosseae, Fu. geosporus, Gl. badium, Cl. etunicatum, Ra. persica. After one year of bait

53
Chapter 1

culturing, the species recovered from the sand dune (in order of decreasing specific
density, see Figure 1) were: Par. occultum, Gl. intraradices, Fu. coronatus, Cl.
claroideum, Cl. etunicatum, Se. constrictum, Fu. mosseae, Di. versiformis, Gl.
microaggregatum, Di. aurantia, Fu. geosporus, Ar. myriocarpa, Ar. trappei. In the field
samples, Se. constrictum was the most abundant species, but not in the trap cultures
Par. occultum had the highest specific density.
Fu. coronatus
Fu. geosporus
Fu. mosseae
1
Gl. badium
Gl. intraradices
0.9 Gl. macrocarpum
Gl. sp. BEV1
Gl. magnicaule
0.8
Gl. sp. BEV2
Specific AMF density (Di)

Gl. microaggregatum
0.7 Gl. microcarpum
Gl. rubiforme
0.6 Se. constrictum
Cl. claroideum
Cl. etunicatum
0.5 En. sp BEV3
Di. aurantia
0.4 Di. clara
Di. versiformis
Di. sp. BEV4
0.3
Ac. scrobiculata
Pa. dominiki
0.2 Pa. franciscana
Ra. persica
Ra. verrucosa
0.1
Sc. calospora
Am. gerdemannii
0 Ar. myriocarpa
Dune 2010 Dune 2011 S. marsh 2010 S. marsh 2011 Ar. trappei
Par. occultum

Figure 1. Specific density (Di) of AMF found in dune and salt marsh at two consecutive years 2010 and
2011 (field-collected soil and bait cultures respectively).

3.7. AM fungal differences in both ecosystems

AMF species and genus richness was similar in both ecosystems (Table 1) with 23
respective 24 out of 30 AMF species and each 11 out of 13 AMF genera detected,
respectively. Ambispora and Entrophospora were the two genera found in the study
area that were not detected in the dune systems but only in the salt marsh system. On
the other hand, Acaulospora and Pacispora species were not found in the salt marsh
while they had been revealed from the dune (Table 1). Other remarkable difference
between both scenarios was the presence of Fu. geosporus and Entrophospora sp.
BEV3 only in the salt marshes. Also Gl. microcarpum, Gl. sp. BEV1, Ra. verrucosa,
Am. gerdemanni and Ar. myriocarpa were only revealed from the salt marshes, while,
beside Ac. scrobiculata, Pa. dominikii and Pa. franciscana, also Gl. magnicaule and Gl.
rubiforme were only detected in the dune but not in the salt marsh.

54
Chapter 1

Table 1. Presence and species richness of AMF at dune and salt marsh directly from field-collected soil
samples in 2010 and from trap cultures in 2011.
Sand Dune Salt Marsh
Trap Trap
In field culture In field culture
AMF species 2010 2011 2010 2011

GLOMERALES
Glomeraceae
Funneliformis coronatus x x x x
Fu. geosporus x x
Fu. mosseae x x x
Glomus badium x x
Gl. intraradices x x x x
Gl. macrocarpum x x
Gl. sp. BEV1 x
Gl. magnicaule x
Gl. sp. BEV2 x x
Gl. microaggregatum x x x x
Gl. microcarpum x
Gl. rubiforme x
Septoglomus constrictum x x x x
Entrophosporaceae
Claroideoglomus claroideum x x x x
Cl. etunicatum x x x
Entrophospora sp. BEV3 x
DIVERSISPORALES
Diversisporaceae
Diversispora aurantia x x
Di. clara x
Di. versiformis x x
Di. sp. BEV4 x x
Acaulosporaceae
Acaulospora scrobiculata x x
Pacisporaceae
Pacispora dominiki x x
Pa. franciscana x x
GIGASPORALES
Racocetraceae
Racocetra persica x x
Ra. verrucosa x
Scutellosporaceae
Scutellospora calospora x x x
ARCHAEOSPORALES
Archaeosporaceae
Ambispora gerdemannii x
Archaeospora myriocarpa x x
Ar. trappei x x
PARAGLOMERALES
Paraglomeraceae
Paraglomus occultum x x
-1 -1
AMF species richness site year 16 16 20 23
Total AMF species richness site-1 23 24
AMF species richness of study 30

55
Chapter 1

4. Discussion

To restore Mediterranean ecosystems, a number of ecophysiological features enhancing


plant performance under drought, salinity and heat, conditions to be exacerbated by
climate change, are frequently looked for (Vallejo et al. 2005). AM fungi are among
some other soil microorganisms that enhance plant performance under stressed
conditions. Knowledge of the plant diversity and their association with AM fungi on
saline areas is of crucial importance for their efficient use in the conservation and
management of endangered ecosystems and agricultural lands affected by salinization.
The data presented in this paper show the AM fungal species found in the rhizosphere
of A. maritimus from the Cabo de Gata Natural Park, Southeast Spain, which it is a
typical Mediterranean ecosystem affected by salinization and desertification. The
vegetal species selected have a high ecological value in Mediterranean ecosystems.
Results obtained by Rodríguez et al. (2005) suggested that A. maritimus could be
considered a useful species in re-vegetation programmes, landscaping and
xerogardening, due to its tolerance to severe water stress and high salinity levels in the
irrigation water. In addition to that, Caravaca et al. (2005) pointed out its capability to
protect the soil from erosion because of the high percentage of stables aggregates found
in the rhizosphere of A. maritimus.
The two sampling areas under study, dune and salt marsh, have in common high levels
of salinity and the ability to accommodate common species of plants, as the target
species of this study A. maritimus, although they differ in many physical, chemical and
biological characteristics. Thus it is not rare to detect common morphotypes of AM
fungi in both zones although also exclusive morphotypes can be found.
Spores were isolated directly from the field samples and from one year old bait cultures,
since it is well known that the use of successive and extended trap cultures detects
higher number of species than extraction of spores directly from field soil (e.g. Ferrol et
al. 2004). Actually, low AMF species richness was often reported for arid and semiarid
ecosystems by extractions of spores from field soil, reflects limitations in the
sporulation patterns under field conditions (e.g. Stutz and Morton 1996; Stutz et al.
2000), but this might depend also on the sampling and isolation effort (Bashan et al.
2007). Nevertheless, we found high AMF species richness at both sites, which was
certainly reasoned in the concomitant extensive analyses of field and bait culture
samples
In both, salt marsh and dune, similar spore morphotypes corresponding to the following
species were recognized: Funneliformis coronatus, Fu. mosseae, Glomus badium, Gl.
intraradices, Gl. macrocarpum, Gl. sp. BEV2, Gl. microaggregatum, Septoglomus
constrictum, Claroideoglomus claroideum, Cl. etunicatum, Diversispora aurantia, Di.
versiformis, Diversispora sp. BEV4, Racocetra persica, Scutellospora calospora,
Archaeospora trappei, Paraglomus occultum.

56
Chapter 1

However, morphotypes assigned to species such as Fu. geosporus, Gl. sp. BEV1, Gl.
microcarpum, Entrophospora sp. BEV3, Ra. verrucosa, Ambispora gerdemannii and
Archaeospora myriocarpa were only found in plants from the salt marsh, while Gl.
magnicaule, Gl. rubiforme, Di. clara, Acaulospora scrobiculata, Pacispora dominiki
and, Pa. franciscana were only detected in the rhizosphere of A. maritimus grown in the
dune.
Since the plant species is not an experimental variable, the reasons for similarities and
differences of the presence of morphotypes should be based on the differential
characteristics between both areas. Specifically, the levels of pH, available phosphorus,
and salinity, have been described as factors that influence the distribution of AM fungi
(Johnson-Green et al. 2001; García and Mendoza 2008).
Similar reasons could also explain the higher density of AMF spores found in the soil
sampled in the A. maritimus rhizosphere of the salt marsh compared to the rhizospheric
samples of the dune at both years of sampling. Another criterion of variability between
the dune and the salt marsh is the spore density of specific species that occurs in some
cases.
Regarding the similarities and discrepancies at the AMF species found in this study,
relevant facts deserve discussion. For instance, it is noteworthy that fungi belonging to
the family Glomeraceae were the predominant sporulators in field and bait conditions
and also presented the highest number of diversity of morphotypes. These species may
be more adapted in adjusting patterns of sporulation to environmental stress conditions
(Jacobson 1997). A similar restriction in species composition to mostly fungi belonging
to Glomus spp. was previously reported in arid and semiarid ecosystems (Jacobson
1997; Stutz et al. 2000).
Previous work indicated that saline soils contain up to 80% of all spores belonging to
one single AMF species, Glomus geosporum (= Fu. geosporus; Carvalho et al. 2001;
Hildebrandt et al. 2001; Landwehr et al. 2002; Grzybowska 2004; Sonjak et al. 2009).
In agreement with the bibliography, Fu. geosporus has been found in the salt marsh,
both at field site and trap culture. However, we found a higher AMF species diversity
than those published and Fu. geosporus was not the most abundant species. In the field
samples, Se. constrictum was the most abundant species in both habitats while in the
bait cultures it only remained predominant in the dune but in the salt marsh Par.
occultum took its place. Both fungi have been widely described in arid and saline
environments: Se. constrictum (Requena et al. 1996; Beena et al. 2001; Kowalchuk et
al. 2002; Ferrol et al. 2004; Rodríguez-Echeverría and Freitas 2006; Wilde et al. 2009;
Hammer et al. 2011), and the genus Paraglomus (Ferrol et al. 2004; Tapia-Goné et al.
2008; Alguacil et al. 2011).
Some Glomeraceae species, Gl. intraradices, Fu. mosseae and Cl. etunicatum for
instance, have been found in saline environments (Aliasgharzadeh et al. 2001;
Landwehr et al. 2002; Tapia-Goné et al. 2008; Sonjak et al. 2009; Wilde et al. 2009;
Camprubí et al. 2010; Yang et al. 2010; Hammer et al. 2011). In the Glomerales order,

57
Chapter 1

three species could not been unequivocally identified, suggesting possible new species;
two of them belonging to the Glomeraceae family, Gl. sp. BEV1 and Gl. sp. BEV2, and
the other to the Entrophosporaceae family, Entrophospora sp. BEV3. The order
Diversisporales gathers the other unidentified species, Diversispora sp. BEV4, and a
new species that have been recently published, Di. clara (Estrada et al. 2011). For
further identification studies, bait cultures have been established to propagate those
unknown fungi.
In this study, only one Acaulosporaceae, Ac. scrobiculata, could be found in the dune,
where the genus Pacispora was very common and widely represented but not in the salt
marsh. Controversially Wilde et al. (2009) found a species of the genus Pacispora, Pa.
scintillans, in the rhizosphere of Puccinelia maritima in Terschelling salt marsh.
Our results also report the occurrence of Racocetra persica among other Gigasporales
morphotypes. This species has been recovered in the Northwestern coast of Italy
(Turrini et al. 2008) and had previously been described from other sand dunes (Koske
and Walker 1985; Blaszkowski and Tadych 1997; Rodríguez-Echeverría and Freitas
2006; Camprubí et al. 2010). Contrary to some results, we did not find any Gigaspora
species in our samples while other authors found several morphotypes (Selvaraj and
Kim 2004; Rodríguez-Echeverría and Freitas 2006; Tapia-Goné et al. 2008). The latter
may be explained according to Turrini et al. (2008) who pointed out that Gigaspora
species are rare in ecosystems with anthropogenic disturbance.
All the information from this study should be considered when designing the AM fungal
inoculum composition for revegetation programs both in natural and agricultural lands
that have become salinized. Schwartz et al. (2006) discussed the use of appropriate
inoculum in restoration programmes and showed that the application of inappropriate
inoculum could result in the upcoming or survival of invasive plant species and the out-
competition of native fungal strains. Thus, the use of autochthonous fungi should be
favoured. Several studies showed the better benefit of native than non native AMF
isolates, which appear to be physiologically and genetically adapted to the stress
conditions of the target environment, in the inoculation strategies used in revegetation
programs of degraded ecosystems (Herrera et al. 1993; Ferrol et al. 2004; Moora et al.
2004; Renker et al. 2004; Oliveira et al. 2005; Querejeta et al. 2006; Alguacil et al.
2011). In addition to that, it may be advisable to use not only one single AMF species
for restoration practices but a mix of a locally adapted AMF community in order to be
able to support a diverse plant cover (Klironomos 2003; Vogelsang et al. 2006).
This study confirms the existence of a rich diversity of AM fungi in the rhizosphere
soils of Spanish saline Mediterranean ecosystems. As inadequate inocula should be
avoided due to possible negative effects on plant species and the plant community, there
is a need to analyze the fungal community associated with target plant species in its
natural environment. In restoration programmes, indigenous inocula should be favoured
over exotic fungi, as the consequences of introduction of non-native fungi on plant
species and plant community structure cannot be precisely predicted. Therefore, this

58
Chapter 1

information should be considered when designing the AM fungal inocula composition


for revegetation programs in order to maximize the potential benefits that these
microorganisms can provide for the establishment of the selected plant species to the
threatened ecosystem.

5. Conclusions

The study reveals a high taxonomic diversity of AM fungi in the rhizosphere of A.


maritimus. The Glomerales order were the predominant sporulators under field and bait
conditions and also presented the highest number of diversity of morphotypes. Common
AM fungi morphotypes both in dunes and salt marshes were found, suggesting the
importance, not only of soil characteristics but the host plant. The genus Pacispora was
very common and widely represented in dunes but not in salt marshes and we could not
identify any spore belonging to Gigaspora genus. One new species, Diversispora clara,
has been recently described and published and four other species are still under study.
The diversity work performed supports the potential for exploiting the natural AM
fungal diversity as inoculum sources to be used in revegetation programmes in arid and
saline ecosystems.

Acknowledgements

This work was financed by two research projects supported by Junta de Andalucía
(Spain). Projects P06-CVI-01876 and P11-CVI-7107.

59
Chapter 1

References

Albaladejo J, Castillo V, Roldán A, Rubio JL, Calvo A (1996) Rehabilitation of


degraded soils by water erosion in semiarid environments. In: Soil degradation
and desertification in Mediterranean Environment. Ediciones Geoforma,
Logroño, pp 265-278
Alcaraz F, Botía M, García R, Ríos S, Rivera D, Robledo A (1997) Flora básica de la
Región de Murcia. Sociedad Cooperativa de Enseñanza Severo Ochoa, Murcia
Alguacil MM, Torres MP, Torrecillas E, Díaz G, Roldán A (2011) Plant type differently
promote the arbuscular mycorrhizal fungi biodiversity in the rhizosphere after
revegetation of a degraded, semiarid land. Soil Biol Biochem 43:167-173
Aliasgharzadeh N, Rastin NS, Towfighi H, Alizadeh A (2001) Occurrence of arbuscular
mycorrhizal fungi in saline soils of the Tabriz Plain of Iran in relation to some
physical and chemical properties of soil. Mycorrhiza 11 :119-122
Allen MF (2007) Mycorrhizal fungi: Highways for water and nutrients in arid soils.
Vadose Zone Journal 6:291-297
Bashan Y, Khaosaad T, Salazar BG, Wiemken A, Oehl F, Vierheilig H (2007) The
mycorrhizal status of Idria columnaris, the boojum tree, an ancient plant from
Baja California. Trees 21:329-335
Beena KR, Arun AB, Raviraja NS, Sridhar KR (2001) Association of arbuscular
mycorrhizal fungi with plants of coastal sand dunes of west coast of India. Trop
Ecol 42:213-222
Blaszkowski J, Tadych M (1997) Scutellospora persica (Glomales, Zygomycetes), an
arbuscular mycorrhizal fungus new to the mycota of Poland. Acta Mycol 28:93-
140
Brundrett M, Melville L, Peterson L (1994) Practical methods in mycorrhizal research.
Mycologue Publications, Ontario, Canada
Camprubí A, Calvet C, Cabot P, Pitet ME, V (2010) Arbuscular mycorrhizal fungi
associated with psammophilic vegetation in Mediterranean coastal sand dunes.
Span J Agric Res 8:96-102
Cantrell IC, Linderman RG (2001) Preinoculation of lettuce and onion with VA
mycorrhizal fungi reduces deleterious effects of soil salinity. Plant Soil 233:269-
281
Caravaca F, Alguacil MM, Torres P, Roldán A (2005) Plant type mediates rhizospheric
microbial activities and soil aggregation in a semiarid Mediterranean salt marsh.
Geoderma 124:375-382
Carvalho LM, Caçador I, Martins-Louçao MA (2001) Temporal and spatial variation of
arbuscular mycorrhizas in salt marsh plants of the Tagus estuary (Portugal).
Mycorrhiza 11:303-309

60
Chapter 1

Dodd JC, Boddington CL, Rodriguez A, Gonzalez-Chavez C, Mansur I (2000)


Mycelium of Arbuscular Mycorrhizal fungi (AMF) from different genera: form,
function and detection. Plant Soil 226:131-151
Entry JA, Rygiewicz PT, Watrud LS, Donnelly PK (2002) Influence of adverse soil
conditions on the formation and function of arbuscular mycorrhizas. Adv
Environ Res 7:123-138
Estrada B, Barea JM, Aroca R, Ruíz-Lozano JM (2012) A native Glomus intraradices
strain from a Mediterranean saline area exhibits salt tolerance and enhanced
symbiotic efficiency with maize plants under salt stress conditions. Plant Soil (In
press) doi:10.1007/s11104-012-1409-y
Estrada B, Palenzuela J, Barea JM, Ruíz-Lozano JM, da Silva GA, Oehl F (2011)
Diversispora clara (Glomeromycetes) - a new species from saline dunes in the
Natural Park Cabo de Gata (Spain). Mycotaxon 118:73-81
Ferrol N, Calvente R, Cano C, Barea JM, Azcón-Aguilar C (2004) Analysing arbuscular
mycorrhizal fungal diversity in shrub-associated resource islands from a
desertification-threatened semiarid Mediterranean ecosystem. Appl Soil Ecol
25:123-133
Francis DF, Thornes JB (1990) Matorral, erosion and reclamation. In: (Eds) Albaladejo
J, Stocking MA, Díaz E (eds) In: Soil Degradation and Rehabilitation in
Mediterranean Environmental Conditions. CSIC, Murcia, Spain, pp 87-115
García IV, Mendoza RE (2008) Relation ships among soil properties, plant nutrition and
arbuscular mycorrhizal fungi-plant symbioses in a temperate grassland along
hydrologic, saline and sodic gradients. FEMS Microbiol Ecol 63:359-371
Geiger F (1973) El Sureste español y los problemas de la aridez. Revista de geografia
7:166-209
Grzybowska B (2004) Arbuscular mycorrhiza of herbs colonizing a salt affected area
near Kraków (Poland). Acta Soc Bot Pol 73:247–253
Hammer E, Nasr H, Pallon J, Olsson P, Wallander H (2011) Elemental composition of
arbuscular mycorrhizal fungi at high salinity. Mycorrhiza 21:117-129
Herrera MA, Salamanca CP, Barea JM (1993) Inoculation of woody legumes with
selected arbuscular mycorrhizal fungi and rhizobia to recover desertified
mediterranean ecosystems. Appl Environ Microbiol 59:129-133
Hewitt EJ (1952) Sand and water culture methods used in the study of plant nutrition.
Technical Communication 22, Farnham Royal; Commonwealth Agricultural
Bureau; Bucks, U.K., pp 547
Hildebrandt U, Janetta K, Ouziad F, Renne B, Nawrath K, Bothe H (2001) Arbuscular
mycorrhizal colonisation of halophytes in Central European salt marshes.
Mycorrhiza 10:175-183
Jacobson KM (1997) Moisture and substrate stability determine VA mycorrhizal fungal
community distribution and structure in arid grassland. J Arid Environ 35:59-75

61
Chapter 1

Jeffries P, Barea JM (2001) Arbuscular Mycorrhiza - a key component of sustainable


plant-soil ecosystems. In: Hock B (ed) The Mycota. Vol. IX. Fungal
Associations. Springer-Verlag, Berlin, Heidelberg, pp 95-113
Johnson-Green P, Kenkel NC, Booth T (2001) Soil salinity and arbuscular mycorrhizal
colonization of Puccinellia nuttalliana. Mycol Res 105:1094-1110
Klironomos JN (2003) Variation in plant response to native and exotic arbuscular
mycorrhizal fungi. Ecology 84:2292-2301
Koske RE, Tessier B (1983) A convenient, permanent slide mounting medium. Myc
Soc America Newsletter 34:59
Koske RE, Walker C (1985) Species of Gigaspora (Endogonaceae) with roughened
outer walls. Mycologia 77:702-720
Kowalchuk GA, De Souza FA, Van Veen JA (2002) Community analysis of arbuscular
mycorrhizal fungi associated with Ammophila arenaria in Dutch coastal sand
dunes. Mol Ecol 11:571-581
Landwehr M, Hildebrandt U, Wilde P, Nawrath K, Toth T, Biro B, Bothe H (2002) The
arbuscular mycorrhizal fungus Glomus geosporum in European saline, sodic
and gypsum soils. Mycorrhiza 12:199-211
Lendínez ML, Marchal FM, Salazar C (2011) Estufio florístico de los medios húmedos
salinos de Andalucía (S. España). Catálogo y análisis de la flora vascular
halófila. Lagascalia 31:77-130
López-Sánchez ME, Honrubia M (1992) Seasonal variation of vesicular-arbuscular
mycorrhizae in eroded soils from southern Spain. Mycorrhiza 2:33-39
Marulanda A, Barea JM, Azcón R (2009) Stimulation of plant growth and drought
tolerance by native microorganisms (AM fungi and bacteria) from dry
environments. Mechanisms related to bacterial effectiveness. J Plant Growth
Regul 28:115-124
Mason E (1928) Note on the presence of mycorrhizae in the roots of salt marsh plants.
New Phytol 27:193-195
Moora M, Öpik M, Sen R, Zobel M (2004) Native arbuscular mycorrhizal fungal
communities differentially influence the seedling performance of rare and
common Pulsatilla species. Funct Ecol 18:554-562
O'Dea ME (2007) Fungal mitigation of soil erosion following burning in a semi-arid
Arizona savanna. Geoderma 138:79-85
Oehl F, Sieverding E, Palenzuela J, Ineichen K, Silva GA (2011) Advances in
Glomeromycota taxonomy and classification. IMA Fungus 2:191-199
Oliveira RS, Vosátka M, Dodd JC, Castro PML (2005) Studies on the diversity of
arbuscular mycorrhizal fungi and the efficacy of two native isolates in a highly
alkaline anthropogenic sediment. Mycorrhiza 16:23-31
Palenzuela J, Barea JM, Ferrol N, Oehl F (2011) Ambispora granatensis, a new
arbuscular mycorrhizal fungus, associated with Asparagus officinalis in
Andalucia (Spain). Mycologia 103:333-340

62
Chapter 1

Palenzuela J, Ferrol N, Boller T, Azcon-Aguilar C, Oehl F (2008) Otospora bareai, a


new fungal species in the Glomeromycetes from a dolomitic shrub land in Sierra
de Baza National Park (Granada, Spain). Mycologia 100:296-305
Pascual JA, Garcia C, Hernandez T, Moreno JL, Ros M (2000) Soil microbial activity
as a biomarker of degradation and remediation processes. Soil Biol Biochem
32:1877-1883
Phillips JM, Hayman DS (1970) Improved procedure of clearing roots and staining
parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of
infection. Trans Br Mycol Soc 55:159-161
Porcel R, Aroca R, Ruiz-Lozano JM (2012) Salinity stress alleviation using arbuscular
mycorrhizal fungi. A review. Agron Sustain Develop 32:181-200
Pozo MJ, Verhage A, García-Andrade J, García JM, Azcón-Aguilar C (2009) Priming
plant defence against pathogens by arbuscular mycorrhizal fungi. In: (eds)
Azcón-Aguilar C, Barea JM, Gianinazzi S, Gianinazzi-Pearson V (eds) In:
Mycorrhizas Functional Processes and Ecological Impact. Springer-Verlag,
Berlin, Heidelberg, pp 123-135
Querejeta JI, Allen MF, Caravaca F, Roldán A (2006) Differential modulation of host
plant δ13C and δ18O by native and nonnative arbuscular mycorrhizal fungi in a
semiarid environment. New Phytol 169:379-387
Renker C, Zobel M, Öpik M, Allen MF, Allen EB, Vosátka M, Rydlová J, Buscot F
(2004) Structure, dynamics, and restoration of plant communities: do arbuscular
mycorrhizae matter? In: Temperton VM, Hobbs, R.J., Nuttle, T., Halle, S. (ed)
Assembly Rules and Restoration Ecology – Bridging the Gap between Theory
and Practice. Washington, DC, USA: Island Press, pp 189-229
Requena N, Jeffries P, Barea JM (1996) Assessment of natural mycorrhizal potential in
a desertified semiarid ecosystem. Appl Environ Microbiol 62:842-847
Requena N, Pérez-Solis E, Azcón-Aguilar C, Jeffries P, Barea JM (2001) Management
of indigenous plant-microbe symbioses aids restoration of desertified
ecosystems. Appl Environ Microbiol 67:495-498
Rillig MC, Mummey DL (2006) Mycorrhizas and soil structure. New Phytol 171:41-53
Rodríguez-Echeverría S, Freitas H (2006) Diversity of AMF associated with
Ammophila arenaria ssp. arundinacea in Portuguese sand dunes. Mycorrhiza
16:543-552
Rodríguez P, Torrecillas A, Morales MA, Ortuño MF, Sánchez-Blanco MJ (2005)
Effects of NaCl salinity and water stress on growth and leaf water relations of
Asteriscus maritimus plants. Environ Exp Bot 53:113-123
Ruiz-Lozano JM, Azcón R (2000) Symbiotic efficiency and infectivity of an
autochthonous arbuscular mycorrhizal Glomus sp. from saline soils and Glomus
deserticola under salinity. Mycorrhiza 10:137-143
Schaede R (1962) Die Pflanzlichen Symbiosen. 2nd ed. Meyer, F.H. Gustav Fischer,
Stuttgart

63
Chapter 1

Schenck NC, Pérez Y (1990) Manual for the Identification of VA Mycorrhizal Fungi.
Synergistic-Publications, Gainesville, Florida
Schwartz MW, Hoeksema JD, Gehring CA, Johnson NC, Klironomos JN, Abbott LK,
Pringle A (2006) The promise and the potential consequences of the global
transport of mycorrhizal fungal inoculum. Ecol Lett 9:501-515
Selvaraj T, Kim H (2004) Ecology of Vesicular-Arbuscular Mycorrhizal (VAM) fungi
in coastal areas of India. Agric Chem Biotechnol 47:71-76
Sieverding E (1991) Vesicular-arbuscular mycorrhiza management in tropical
agrosystems. Technical Cooperation (GTZ). Eschborn. Friedland, Bremer,
Rossdorf, TZ-Verlagsgesellschaft, Germany
Slezack S, Dumas-Gaudot E, Paynot M, Gianinazzi S (2000) Is a fully established
arbuscular mycorrhizal symbiosis required for bioprotection of Pisum sativum
roots against Aphanomyces euteiches? Mol Plant-Microbe Interact 13:238-241
Smith SE, Read DJ (2008) Mycorrhizal Symbiosis, 3rd Ed. Elsevier, Academic Press,
New York
Sonjak S, Udovic M, Wraber T, Likar M, Regvar M (2009) Diversity of halophytes and
identification of arbuscular mycorrhizal fungi colonising their roots in an
abandoned and sustained part of Secovlje salterns. Soil Biol Biochem 41:1847-
1856
Stutz JC, Copeman R, Martín CA, Morton JB (2000) Patterns of species composition
and distribution of arbuscular mycorrhizal fungi in arid regions of southwestern
North America and Namibia. Afr Can J Bot 78:237-245
Stutz JC, Morton JB (1996) Successive pot cultures reveal high species richness of
arbuscular endomycorrhizal fungi in arid ecosystems. Can J Bot 74:1883-1889
Tapia-Goné J, Ferrera-Cerrato R, Varela-Fregoso L, Rodríguez Ortiz JC, Lara Mireles J,
Soria Colunga JC, Cuellar Torres H, Tiscareño Iracheta MA, Cisneros Almazán
R (2008) Caracterización e identificación morfológica de hongos formadores de
micorriza arbuscular, en cinco suelos salinos del estado de San Luis Potosí,
México. Revista Mexicana de Micología 26:1-7
Toth R, Miller RM, Jarstfer A, Alexander A, Bennet EL (1991) The calculation of
intraradical fungal biomass from percent colonisation in vesicular-arbuscular
mycorrhizae. Mycologia 83:553-558
Trappe JM (1981) Mycorrhizae and Productivity of Arid and Semiarid Rangelands.
Academic Press, New York
Turrini A, Avio L, Bedina S, Giovannetti M (2008) In situ collection of endangered
arbuscular mycorrhizal fungi in a Mediterranean UNESCO Biosphere Reserve.
Biodivers Conserv 17:643-657
Vallejo VR, Aronson J, Pausas JG, Cortina J (2005) Restoration of Mediterranean
woodlands. In: Andel JV, Aronson, JJ. (ed) Restoration ecology: the new
frontier. Blackwell Publishing, Oxford, United Kingdom, pp 193-207

64
Chapter 1

Vallejo VR, Bautista S, Cortina J (1999) Restoration for soil protection after
disturbances. In: Trabaud L (ed) Life and Environment in the Mediterranean.
Advances in Ecological Sciences. WIT Press, Wessex, pp 301-343
van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R,
Boller T, Wiemken A, Sanders IR (1998) Mycorrhizal fungal diversity
determines plant biodiversity, ecosystem variability and productivity. Nature
396:69-72
van der Heijden MGA, Streitwolf-Engel R, Riedl R, Siegrist S, Neudecker A, Ineichen
K, Boller T, Wiemken A, Sanders IR (2006) The mycorrhizal contribution to
plant productivity, plant nutrition and soil structure in experimental grassland.
New Phytol 172:739-752
Vogelsang KM, Reynolds HL, Bever JD (2006) Mycorrhizal fungal identity and
richness determine the diversity and productivity of a tallgrass prairie system.
New Phytol 172:554-462
Wilde P, Manal A, Stodden M, Sieverding E, Hildebrandt U, Bothe H (2009)
Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt
marshes. Environ Microbiol 11:1548-1561
Yang C, Hamel C, Schellenberg MP, Pérez JC, Berbara RL (2010) Diversity and
functionality of arbuscular mycorrhizal fungi in three plant communities in
semiarid grasslands National Park, Canada. Microb Ecol 59:724-733

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CAPÍTULO 2
CHAPTER 2

Diversispora clara (Glomeromycetes) – a new species from saline dunes


in the Natural Park Cabo de Gata (Spain)

Reprinted from Mycotaxon, 2011, 118: 73-81 (Estrada B, Palenzuela J, Barea J.M,
Ruiz-Lozano J.M, da Silva G.A and Oehl F.)
Capítulo 2

Diversispora clara (Glomeromycetes) – una nueva especie de dunas


salinas del Parque Natural Cabo de Gata (España)

Resumen

En las dunas del Parque Natural Cabo de Gata (Almería, Andalucía, sur de España) se
encontró una nueva especie de Diversispora (Glomeromycetes) en la rizosfera de
Asteriscus maritimus, una especie vegetal especialmente adaptada a ambientes salinos.
La nueva especie de hongo formador de micorrizas arbusculares forma esporas blancas
brillantes que tienen un tamaño de 79-130 × 75-125 micras y tienen una pared que
consta de tres capas. La hifa sustentoria es como la típica de muchas Diversispora spp.,
de pared delgada, hialina y cilíndrica (o rara vez constreñida) y flexible y frágil por
debajo de los septos que separan el contenido de esporas y de hifas. Los septos se
forman regularmente en la base de las esporas o, con menor frecuencia, en la hifa
sustentoria a corta distancia de la base de las esporas. El análisis filogenético del ITS y
parte del gen ribosómico 28S confirman que D. clara forma un clado independiente y
monofilético dentro de Diversispora.

Palabras clave: Glomus, Glomeromycota, Europa, ambientes extremos, ADNr

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Diversispora clara (Glomeromycetes) – a new species from saline dunes


in the Natural Park Cabo de Gata (Spain)

Beatriz Estrada1, Javier Palenzuela1, José-Miguel Barea1, Juan Manuel Ruiz-


Lozano1, Gladstone Alves da Silva2 & Fritz Oehl3*

1
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación
Experimental del
Zaidín, CSIC, Profesor Albareda 1, 18008 Granada, Spain
2
Departamento de Micologia, CCB, Universidade Federal de Pernambuco,
Av. Prof. Nelson Chaves s/n, Cidade Universitária, 50670-420, Recife, PE, Brazil
3
Federal Research Institute Agroscope Reckenholz-Tänikon ART,
Organic Farming Systems, Reckenholzstrasse 191, CH-8046 Zürich, Switzerland

*Correspondence to: fritz.oehl@art.admin.ch

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Abstract

A new species of Diversispora (Glomeromycetes) was found in saline sand dunes of the
Natural Park Cabo de Gata (Almería, Andalucía, Southern Spain) in the rhizosphere of
Asteriscus maritimus, a plant species especially adapted to saline environments. The
new fungal species forms brilliant white spores that are 79-130 × 75-125 μm and have
one wall consisting of three layers. The subtending hyphae are, as typical for many
Diversispora spp., thin-walled, hyaline, and cylindrical (or rarely constricted) and
flexible and fragile below the septa separating the spore and hyphal contents. The septa
form regularly at the spore bases or, less frequently, in subtending hyphae at short
distances from the spore base. Phylogenetic analyses of the ITS and partial 28S
ribosomal gene confirm that D. clara forms a monophyletic, independent clade within
Diversispora.

Key words: Glomus, Glomeromycota, Europe, extreme environments, rDNA.

Introduction

During recent studies on arbuscular mycorrhiza (AM) fungal diversity in sand dune
systems of the Cabo de Gata Natural Park in Almería (Spain), a brilliant-white new
glomeromycotean fungus was recovered from the rhizosphere of Asteriscus maritimus,
a plant species characteristic of saline Mediterranean environments with elevated soil
electrical conductivity. The new fungus formed spores in AM fungal bait cultures
predominantly in the Asteriscus maritimus rhizosphere. The aims of the present study
were to analyze this particular fungus applying combined morphological and molecular
tools and to describe its characteristics.

Material and methods

Soil and plant sampling

Soil samples were taken in February 2010 from the rhizosphere of ten Asteriscus
maritimus plants growing in a natural sand dune system of the Natural Park Cabo de
Gata in Almería (Andalucía, Spain). The site is located at 36°44´41´´N 02°07´26´´W.
The samples, air-dried in the laboratory, were used to analyze selected chemical soil
parameters (pH, soil organic carbon, total nitrogen, soil electrical conductivity) and
spore populations The ten plants and the surrounding rhizosphere soil were also
extracted and used as bait cultures for propagation of AM fungal communities
indigenous to the natural sand dune system. At the site, the soil was sandy and with pH

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(H2O) 8.2, organic carbon 15.3 g kg-1, total nitrogen 1.9 g kg-1, available P 27.0 mg kg-1,
and soil electrical conductivity 0.5 dS m-1.

AM fungal bait cultures

Bait cultures were established and maintained as described in Palenzuela et al. (2008,
2011) by transplanting 10 Asteriscus maritimus plants with their rhizosphere soils into 1
L pots and transferring them to the greenhouse at EEZ in Granada immediately after
sampling. The pots were irrigated three times per week and fertilized every four weeks
with Long-Aston nutrient solution (Hewitt 1966). Pure cultures of the new fungus were
initiated in a mixed-culture of three Allium porrum L. and three Hieracium pilosella L.
plantlets in 750 mL pots grown together at three locations in the pots. The plant
rhizosphere was inoculated with 20 spores per pot and pot location. A sterile mixture of
Terragreen (American aluminum oxide, Oil Dry US special, type III R; Lobbe
Umwelttechnik Iserlohn, Germany) and Loess (mixture 3:1; with pH-KCl 6.2; organic
carbon 0.3%; available P (Na-acetate) 2.6 mg kg-1; available K (Na-acetate) 350 mg kg-1
was chosen as culture substrate (Oehl et al. 2002). So far, pure cultures of the new
fungus have not been obtained.

Morphological analyses

AM fungal spores were separated from the soil samples by a wet sieving process
(Sieverding 1991). The morphological spore characteristics and their subcellular
structures were described from a specimen mounted in: polyvinyl alcohol-lactic
acidglycerol (PVLG; Koske and Tessier 1983); a mixture of PVLG and Melzer’s
reagent (Brundrett et al. 1994); a mixture of lactic acid to water at 1:1; Melzer’s reagent;
and water (Spain 1990). The spore structure terminology follows Oehl et al. (2003,
2005, 2011a) for species with glomoid or diversisporoid spore formation. Photographs
(Figs. 1-10) were taken with a Leica DFC 290 digital camera on a Leitz Laborlux S
compound microscope using Leica Application Suite Version V 2.5.0 R1 software.
Specimens mounted in PVLG and the PVLG+Melzer’s mixtures were deposited at the
herbaria Z+ZT (ETH Zurich, Switzerland), GDA-GDAC (University of Granada,
Spain), and URM (Federal University of Pernambuco, Recife).

Molecular analyses

Five spores isolated from the trap cultures were surface-sterilized with chloramine T
(2%) and streptomycin (0.02%) (Mosse 1962) and crushed with a sterile disposable
micropestle in 40 μL milli-Q water (Ferrol et al. 2004). PCRs of the crude extracts were
obtained in an automated thermal cycler (Gene Amp PCR System 2400, Perkin-Elmer,
Foster City, California) with a pureTaq Ready-To-Go PCR Bead (Amersham

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Biosciences Europe GmbH, Germany) following manufacturer’s instructions with 0.4


μM concentration of each primer. A two-step PCR amplified the partial SSU, ITS1,
5.8S, ITS2 and partial LSU rDNA ribosomal fragment using the SSUmAf/LSUmAr and
SSUmCf/LSUmBr primers consecutively (Krüger et al. 2009). The second PCR
products were separated electrophoretically on 1.2% agarose gels stained with Gel
Red™ (Biotium Inc., Hayward, CA, U.S.A.) and viewed by UV illumination. The band
of the expected size was excised with a scalpel and the amplified DNA was isolated
from the gel with the QIAEX II Gel Extraction kit (QIAGEN, Valencia, CA, USA),
cloned into the PCR2.1 vector (Invitrogen, Carlsbard, CA, USA), and transformed into
one shot TOP10 chemically competent Escherichia coli cells. After plasmid isolation
from transformed cells, cloned DNA fragments were sequenced with vector primers
(White et al. 1990) in both directions by Taq polymerase cycle sequencing on an
automated DNA sequencer (Perkin-Elmer ABI Prism 373). Sequence data were
compared to gene libraries (EMBL and GenBank) using BLAST (Altschul et al. 1990).
The new sequences were deposited in the EMBL database under the accession numbers
FR873629-FR873633.

PHYLOGENETIC ANALYSES: The AM rDNA ITS1+5.8S +ITS2 fungal sequences


were aligned in ClustalX (Larkin et al. 2007) and edited with BioEdit (Hall 1999) to
obtain a final alignment with Acaulospora laevis Gerd. & Trappe and A. lacunosa J.B.
Morton as outgroups. Prior to phylogenetic analysis, the model of nucleotide
substitution was estimated using Topali 2.5 (Milne et al. 2004). Bayesian (two runs over
1 × 106 generations with a burn in value of 2500) and maximum likelihood (1000
bootstrap) analyses were performed in MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003)
and PhyML (Guindon & Gascuel 2003) respectively, launched from Topali 2.5, using
the GTR + G model. Neighbor-joining (established with the model cited above) and
maximum parsimony analyses were performed using PAUP*4b10 (Swofford 2003)
with 1000 bootstrap replications.

Results

Diversispora clara Oehl, B. Estrada, G.A. Silva & Palenz., sp. nov. Figs 1-10
MycoBank MB 561583
Sporae albae, 79–130 × 75–125 μm, tunica tribus stratis, 4.3–8.4 μm. Stratum
exterior hyalinum; stratum medium, album ad rarum ochreo-album, 2.8–5.4 μm
crassum; stratum interius album. Hypha adhaerenta, 8–13 μm in diametrum.
Tunica hyphae 1.0–1.8 μm crassa. Strata medium interiusque septo porum
occludentes. Strata exterior mediumque flava colorantes Melzeri.
TYPE: Spain. Andalucía, Almería, Cabo de Gata Natural Park, sand dune, from
the rhizosphere of Asteriscus maritimus (L.) Less. (Asteraceae), isolation date

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February 2011, by B. Estrada (holotype, ZT Myc 3796 [permanent slide 20-


2001]; isotypes, ZT Myc 3797 [permanent slides 20-2003 to 20-2012], GDA-
GDAC [permanent slides 20-2013 to 20-2018], URM [permanent slides 20-
2019, 20-2020]).
ETIMOLOGY: clara (Latin = clear, bright, brilliant, light), referring to the
brilliant white spores.

FIGS 1-10. Diversispora clara: 1-4. Uncrushed spores with a triple-layered spore wall (swl1-3), a single,
cylindrical subtending hypha (sh), and a septum (sp) arising directly or at some distance from the base.
Figs 5–6. Spores crushed to show triple layered spore wall (swl1-3). FIGS 7-8. Spore wall structure in
PVLG + Melzer’s reagent. The swl1 or swl2 (or both) stain light to dark yellow in Melzer’s, while no
such staining reaction is seen on swl3. However, the yellow stain is usually most noticeable in the spore
cell contents. FIG. 9. Septum at spore base formed by swl2 (swl3 is not visible in uncrushed spore). FIG.
10. Septum at spore base formed by swl3.

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SPORES (Figs 1-4) singly formed in rhizosphere soils, terminally on subtending


hyphae, globose (80-110 μm diam.) to subglobose (79-130 × 75-125 μm), one-walled,
brilliant to creamy white.

SPORE WALL 4.3-8.4 μm thick, three-layered (swl1, swl2, swl3; Figs 5-7);
outer layer (swl1) hyaline, 0.8-1.5 μm thick, evanescent and thus often absent in mature
spores; second layer (swl2) bright to (rarely) creamy white, laminated, 2.8-5.4 μm thick
(Fig. 7) in uncrushed spores (sometimes ≤ 6.6 μm in crushed spores when pressure is
applied on the cover slip); inner layer (swl3) brilliant white, 0.7-1.5 μm thick (usually
tightly adherent to swl2 and difficult to observe when < 1.0 μm; see Figs 5-6); both
swl1 and swl2 generally light to dark yellow in Melzer’s reagent (Figs 7-8, with spore
cell contents often a more intense yellow than the surrounding cell wall layers (Fig. 8).

SUBTENDING HYPHAE (sh) generally singly on spores; brilliant white (Figs


1-3), cylindrical or (rarely) slightly constricted at the spore base, 4.0-8.0 μm broad
tapering to 3.2-6.1 μm within 100 μm of spore base, although the distance may appear
shorter (4.0-10(-25) μm) because the sh walls taper from 1.0-1.8 μm thick to 0.6-1.2 μm
within the first 10 μm from the base causing the flexible fragile portion to break from
the mature spore (Figs 3, 4, 9) at a point where the septum separates the spore contents
from the hyphal contents. Spore pores at the spore bases or in the sh normally closed by
a septum (Figs 1-2, 4) arising from swl2 (Fig. 9), swl3 (Fig. 10), or both layers (not
shown); pores rarely open (Fig. 3).

DISTRIBUTION: Known only in the natural sand dune system of the Cabo de
Gata Natural Park in Andalucía in the rhizosphere of Asteriscus maritimus.

MOLECULAR ANALYSES: Phylogenies derived from ITS (Fig. 11) and 28S
(data not shown) rDNA analyses cluster the new fungus within the Diversisporaceae in
a well-separated clade adjacent to several other Diversispora species, Otospora bareae,
and Tricispora nevadensis (Oehl et al. 2011b).

Discussion

Our morphological analyses, in particular those of the subtending hyphae and


spore bases, clearly support the new fungus in Diversispora, with many characters
identical to those of other Diversispora species (e.g., D. spurca, D. eburnea; see Oehl et
al. 2011a,b) such as the thin-walled, hyaline, cylindrical (or rarely constricted)
subtending hyphae that appear flexible and fragile behind the septum that closes the
pore at the spore base or in short distance from the base. In Diversispora species with
pigmented spores, the subtending hyphae regularly change color conspicuously,
becoming hyaline to white behind the septum (Oehl et al. 2011a). This color change,

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however, was not confirmed for D. clara, since the new fungus generally does not form
pigmented spores.
Molecular analyses confirm the species as new: in the phylogenetic tree, D.
clara clusters in a independent, monophyletic clade within a polyphyletic Diversispora.
These sequence analyses also confirm unequivocally using morphology to identify
diversisporoid species within the Glomeromycota (Oehl et al. 2011a,c).
Including D. clara, there are now 14 Diversispora spp. known in the
Glomeromycetes (Oehl et al. 2011a,b). The 9 species that form significantly pigmented,
yellow brown to brown or orange to orange brown spores are D. arenaria (Błaszk. et
al.) Oehl et al., D. aurantia (Błaszk. et al.) C. Walker & A. Schüssler, D. epigaea (B.A.
Daniels & Trappe) C. Walker & A. Schüssler, D. insculpta (Błaszk.) Oehl et al., D.
przelewicensis (Błaszk.) Oehl et al., D. pustulata (Koske et al.) Oehl et al., D. tenera
(P.A. Tandy) Oehl et al., D. trimurales (Koske & Halvorson) C. Walker & A. Schüssler,
and D. versiformis (P. Karst.) Oehl et al. (Oehl et al. 2011a,b). Diversispora celata C.
Walker et al. forms triple-layered, ochre to ivory to pinkish cream spores (Gamper et al.
2009). Only D. spurca (C.M. Pfeiff. et al.) C. Walker & A. Schüssler, D. Eburnea (L.J.
Kenn. et al.) C. Walker & A. Schüssler, and D. gibbosa (Błaszk.) Błaszk. & Kovács
form hyaline to subhyaline spores that are, however, never brilliantwhite as observed for
D. clara. Moreover, D. spurca and D. eburnea have bilayered spore walls (Kennedy et
al. 1999), D. gibbosa has a five-layered wall (Błaszkowski 1997), and D. clara has a
three-layered wall.
In the past, large-spored or sporocarpic AM fungi were described from sand
dune systems, such as Gigaspora, Scutellospora, Pacispora or Glomus species that
were generally easy to isolate and recognize from field samples (Koske & Gemma
1995, Gemma et al. 1989, Błaszkowski 1994). Likewise in the current Cabo de Gata
Natural Park sand dune system, where Funneliformis coronatus (Giovann.) C. Walker
& A. Schüssler, F. mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schüssler,
Scutellospora calospora (T.H. Nicolson & Gerd.) C. Walker & F.E. Sanders, Racocetra
persica (Koske & C. Walker) Oehl et al., and Glomus macrocarpum Tul & C. Tul. were
identified (Estrada, unpublished). When sand dune AM fungal communities were
maintained and reproduced in bait cultures, small-spored Glomus spp. were also
sometimes detected (e.g. Błaszkowski et al. 2009a,b, 2010). It was supposed that
species with small, quickly degrading spores were difficult to recover or identify only
from field samples. This might also be true for D. clara, even though its laminated wall
structure is clearly persistent. It will be interesting to see whether future taxonomists
will be able to identify the new species directly from field samples, now that the
existence and morphology of this unique, brilliant-white, conspicuous but small-spored
species is known. Morphological spore and molecular root and spore analyses will
hopefully tell us more about the ecology and biogeography of this fungus that is thus far
known only from a single Asteriscus maritimus rhizosphere in the Natural Park Cabo de
Gata of Almería in southern Spain.

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FIG. 11. Diversisporaceae. ITS rDNA-based phylogenetic tree rooted by Acaulospora laevis and A.
lacunosa. Sequences are labeled with database accession numbers. Support values are from neighbor-
joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian analyses. Diversispora
clara sequences are in bold. Only topologies with ≥ 50% bootstrap values are shown. (Consistency Index
= 0.72; Retention Index = 0.84).

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Acknowledgments

This study has been supported by the Junta de Andalucía (Spain), project P06-CVI-
01876 and by the Swiss National Science Foundation (SNSF; Project
315230_130764/1). We acknowledge the valuable comments on the manuscript and
revisions of Dr. Bruno Tomio Goto (Universidade Federal do Rio Grande do Norte,
UFRN, Natal, Brazil), Dr. Iván Sánchez-Castro (INRA, Dijon, France) and PD Dr.
Ewald Sieverding (University of Hohenheim, Germany) and appreciate the corrections
by Shaun Pennycook, Nomenclatural Editor, and suggestions by Lorelei L. Norvell,
Editor-in-Chief.

Literature cited

Altschul SF, Gish W, Miller W, Myers EW, Lipton DJ. 1990. Basic local alignment
search tool. J. Mol.Biol. 215: 403–410. http://dx.doi.org/10.1006/jmbi.1990.9999
Błaszkowski J. 1994. Arbuscular fungi and mycorrhizae (Glomales) of the Hel
Peninsula, Poland.Mycorrhiza 5, 71–88. http://dx.doi.org/10.1007/BF00204022
Błaszkowski J. 1997. Glomus gibbosum, a new species from Poland. Mycologia 89(2):
339–345. http://dx.doi.org/10.2307/3761092
Błaszkowski J, Ryszka P, Oehl F, Koegel S, Wiemken A, Kovács GM, Redecker D.
2009a. Glomusachrum and G. bistratum, two new species of arbuscular mycorrhizal
fungi (Glomeromycota).Botany 87: 260–271. http://dx.doi.org/10.1139/B08-138
Błaszkowski J, Kovács GM, Balázs TK. 2009b. Glomus perpusillum, a new arbuscular
mycorrhizalfungus. Mycologia 101(2): 247–255. http://dx.doi.org/10.3852/08-087
Błaszkowski J, Wubet T, Harikumar VS, Ryszka P, Buscot F. 2010. Glomus indicum, a
new arbuscularmycorrhizal fungus. Botany 88: 132–143.
http://dx.doi.org/10.1139/B09-104
Brundrett M, Melville L, Peterson L. 1994. Practical methods in mycorrhizal research.
MycologuePublications, University of Guelph, Guelph, Ontario, Canada.
Ferrol N, Calvente R, Cano C, Barea JM, Azcón-Aguilar C. 2004. Analyzing arbuscular
mycorrhizalfungal diversity in shrub-associated resource islands from a
desertification-threatened semiaridMediterranean ecosystem. Appl. Soil Ecol. 25:
123–133.http://dx.doi.org/10.1016/j.apsoil.2003.08.006
Gamper HA, Walker C, Schüßler A. 2009. Diversispora celata sp. nov.: molecular
ecology andphylotaxonomy of an inconspicuous arbuscular mycorrhizal fungus.
New Phytol. 182(2):495–506. http://dx.doi.org/10.1111/j.1469-8137.2008.02750.x
Gemma JN, Koske RE, Carreiro MM. 1989. Seasonal dynamics of five species of VA
fungi in a sand dune. Mycol. Res. 92: 27–32 http://dx.doi.org/10.1016/S0953-
7562(89)80072-3

79
Chapter 2

Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm to estimate large
phylogenies by maximum likelihood. Systematic Biol. 52: 696–704.
http://dx.doi.org/10.1080/10635150390235520
Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and
analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41: 95–98.
Hewitt EJ. 1966. Sand and water culture methods used in the study of plant nutrition.
Farnham Royal, Farnham, England: Commonwealth Agricultural Bureau. 547 p.
Kennedy LJ, Stutz JC, Morton JB. 1999. Glomus eburneum and G. luteum, two new
species of arbuscular mycorrhizal fungi, with emendation of G. spurcum. Mycologia
91(6): 1083–1093. http://dx.doi.org/10.2307/3761638
Koske R., Gemma JN. 1995. Scutellospora hawaiiensis (Gigasporaceae): a new species
of arbuscular mycorrhizal fungi from Hawaii. Mycologia 87: 679–684.
http://dx.doi.org/10.2307/3760811
Koske RE, Tessier B. 1983. A convenient, permanent slide mounting medium. Mycol.
Soc. Am. Newsl. 34: 59.
Krüger M, Stockinger H, Krüger C, Schüßler A. 2009. DNA-based species-level
detection of arbuscular mycorrhizal fungi: one PCR primer set for all AMF. New
Phytol. 183(1): 212–223. http://dx.doi.org/10.1111/j.1469-8137.2009.02835.x
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H,
Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG.
2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.
http://dx.doi.org/10.1093/bioinformatics/btm404
Milne I, Wright F, Rowe G, Marshal DF, Husmeier D, McGuire G. 2004. TOPALi:
Software for Automatic Identification of Recombinant Sequences within DNA
Multiple Alignments.Bioinformatics 20: 1806–1807.
http://dx.doi.org/10.1093/bioinformatics/bth155
Mosse B. 1962. Establishment of vesicular-arbuscular mycorrhiza under aseptic
conditions. J. Gen. Microbiol. 27: 509–520.
Oehl F, Wiemken A, Sieverding E. 2002. Glomus caesaris, a new arbuscular
mycorrhizal fungus from the Kaiserstuhl in Germany. Mycotaxon 84: 379–385.
Oehl F, Wiemken A, Sieverding E. 2003. Glomus aureum, a new sporocarpic species in
the Glomales from European grasslands. J. Appl. Bot. 77: 111–115.
Oehl F, Redecker D, Sieverding E. 2005. Glomus badium, a new sporocarpic arbuscular
Mycorrhizal fungal species from European grasslands of higher soil pH. J. Appl.
Bot. Food Qual. 79: 38–43.
Oehl F, Silva GA, Goto BT, Sieverding E. 2011a. Glomeromycota: three new genera,
and glomoid species reorganized. Mycotaxon 116: 75–120.
http://dx.doi.org/10.5248/116.75
Oehl F, Silva GA, Sánchez-Castro I, Goto BT, Maia LC, Vieira HEE, Barea JM,
Sieverding E, Palenzuela J. 2011b. Revision of Glomeromycetes with

80
Chapter 2

entrophosporoid and glomoid spore formation, with three genera nova. Mycotaxon
117: 297–316, http://dx.doi.org/10.5248/117.297
Oehl F, Sieverding E, Palenzuela J, Ineichen K, Silva GA. 2011c. Advances in
Glomeromycota taxonomy and classification. IMA Fungus 2: 191–199.
http://dx.doi.org/10.5598/imafungus.2011.02.02.10
Palenzuela J, Ferrol N, Boller T, Azcón-Aguilar C, Oehl F. 2008. Otospora bareai, a
new fungal species in the Glomeromycetes from a dolomitic shrub-land in the
National Park of Sierra de Baza (Granada, Spain). Mycologia 100: 296–305.
http://dx.doi.org/10.3852/mycologia.100.2.296
Palenzuela J, Barea JM, Ferrol N, Oehl F. 2010. Entrophospora nevadensis, a new
arbuscular mycorrhizal fungus, from Sierra Nevada National Park (southeastern
Spain). Mycologia 102: 624–632, http://dx.doi.org/10.3852/09-145
Palenzuela J, Barea JM, Ferrol N, Oehl F. 2011. Ambispora granatensis, a new
arbuscular Mycorrhizal fungus, associated with Asparagus officinalis in Andalucía
(Spain). Mycologia 103: 333–340. http://dx.doi.org/10.3852/09-146
Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under
mixed models. Bioinformatics 19: 1572–1574.
http://dx.doi.org/10.1093/bioinformatics/btg180
Sieverding E. 1991. Vesicular-arbuscular mycorrhizal management in tropical
agrosystems. Technical Cooperation (GTZ). Eschborn. Friedland, Bremer,
Rossdorf, TZ-Verlagsgesellschaft. Germany.
Sieverding E, Oehl F. 2006. Revision of Entrophospora and description of Kuklospora
and Intraspora, two new genera in the arbuscular mycorrhizal Glomeromycetes. J.
Appl. Bot. Food Qual. 80:69–81.
Spain JL. 1990. Arguments for diagnoses based on unaltered wall structures.
Mycotaxon 38: 71–76.
Swofford DL. 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and other
methods). Sinauer Associates, Sunderland, Massachusetts.
White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenetics. 315–322, in: MA Innis et al. (eds).
PCR protocols: a guide to methods and applications. Academic Press, San Diego,
California.

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CAPÍTULO 3
CHAPTER 3

A native Glomus intraradices strain from a Mediterranean saline area


exhibits salt tolerance and enhanced symbiotic efficiency with maize
plants under salt stress conditions

Reprinted from Plant and Soil, In press (2012), doi:10.1007/s11104-012-1409-y


(Estrada B, Barea J.M, Aroca R and Ruiz-Lozano J.M)

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Capítulo 3

Un aislado de Glomus intraradices nativo de un área Mediterránea


salina muestra tolerancia a la salinidad y elevada eficiencia simbiótica
en plantas de maíz bajo condiciones de estrés salino

Resumen

Los hongos micorrícicos arbusculares (MA) se ha visto que existen de forma natural en
ambientes salinos y se ha sugerido que las diferencias en el comportamiento de estos
hongos y su eficiencia puede ser debida al origen y la adaptación del hongo MA. Estos
resultados invitan a buscar especies de MA aisladas en ambientes salinos y comparar
sus mecanismos de tolerancia a la salinidad con aquellas especies de áreas no-salinas.
Para ello un aislado de G. intraradices (Gi CdG) perteneciente a una región con graves
problemas de salinidad y afectada por la desertificación se ha comparado con un aislado
de colección de la misma especie, que se usa habitualmente como hongo modelo. Un
experimento in vitro comprobó la capacidad de ambos hongos MA de crecer bajo
niveles crecientes de salinidad en el medio y un experimento in vivo comparó su
eficiencia simbiótica con las plantas de maíz cultivadas bajo condiciones de estrés
salino. El aislado Gi CdG se desarrolló mejor bajo condiciones de salinidad e indujo
considerablemente la expresión de los genes GintBIP, Gint14-3-3 y GintAQP1, si bien
mostró una inducción más baja del gen GintSOD1 que la cepa de colección G.
intraradices. El aislado Gi CdG también estimuló el crecimiento de las plantas de maíz
más que la cepa de colección bajo dos niveles de salinidad. La mayor eficiencia
simbiótica de Gi CdG fue corroborada por el aumento de la eficiencia del fotosistema II
y la conductancia estomática y la menor pérdida de electrolitos mostrada por plantas de
maíz bajo las diferentes condiciones probadas. La mayor tolerancia a la salinidad y la
eficiencia simbiótica exhibida por el aislado Gi CdG en comparación con el G.
intraradices de colección puede ser debido a una adaptación del hongo a los ambientes
salinos. Tal adaptación puede estar relacionada con la importante inducción de la
expresión de los genes que codifican para chaperonas o para acuaporinas del hongo. El
presente estudio muestra que los hongos MA aislados en zonas afectadas por salinidad
pueden ser una herramienta poderosa para mejorar la tolerancia de los cultivos a
condiciones de estrés salino.

Palabras clave: Adaptación, micorriza arbuscular, monoxénico, salinidad, eficiencia


simbiótica

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Chapter 3

A native Glomus intraradices strain from a Mediterranean saline area


exhibits salt tolerance and enhanced symbiotic efficiency with maize
plants under salt stress conditions

Beatriz Estrada, José Miguel Barea, Ricardo Aroca and


Juan Manuel Ruiz-Lozano*

Departamento de Microbiología del Suelo y Sistemas Simbióticos. Estación


Experimental del Zaidín (CSIC). Profesor Albareda nº 1, 18008 Granada, Spain.

* Corresponding author: Dr. Juan Manuel Ruiz-Lozano.


e-mail: juanmanuel.ruiz@eez.csic.es
Telph. + 34 958 181600
Fax: + 34 958 129600

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Abstract

Aims:
Arbuscular mycorrhizal (AM) fungi have been shown to occur naturally in saline
environments and it has been suggested that differences in fungal behaviour and
efficiency can be due to the origin and adaptation of the AM fungus. These findings
invite to look out for AM fungal species isolated in saline environments and compare
their salt-tolerance mechanisms with those of species living in non-saline areas.
Methods:
A fungal strain of G. intraradices (Gi CdG) isolated from a region with serious
problems of salinity and affected by desertification, has been compared with a
collection strain of the same species, used as a model fungus. An in vitro experiment
tested the ability of both AM fungi to grow under increasing salinity and an in vivo
experiment compared their symbiotic efficiency with maize plants grown under salt
stress conditions.
Results:
The isolate Gi CdG developed better under saline conditions and induced considerably
the expression of GintBIP, Gint14-3-3 and GintAQP1 genes, while it showed a lower
induction of GintSOD1 gene than the collection G. intraradices strain. The isolate Gi
CdG also stimulated the growth of maize plants under two levels of salinity more than
the collection strain. The higher symbiotic efficiency of Gi CdG was corroborated by
the enhanced efficiency of photosystem II and stomatal conductance and the lower
electrolyte leakage exhibited by maize plants under the different conditions assayed.
Conclusions:
The higher tolerance to salinity and symbiotic efficiency exhibited by strain Gi CdG as
compared to the collection G. intraradices strain may be due to a fungal adaptation to
saline environments. Such adaptation may be related to the significant up-regulation of
genes encoding chaperones or genes encoding aquaporins. The present study remarks
that AM fungi isolated from areas affected by salinity can be a powerful tool to enhance
the tolerance of crops to saline stress conditions.

Key words: adaptation, arbuscular mycorrhiza, monoxenic culture, salinity, symbiotic


efficiency

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Introduction

Soil salinity exists naturally on Earth, but inadequate cultivation practices,


mainly due to excess irrigation (Zhu 2001; Tester and Davenport 2003; Flowers 2004)
have also exacerbated growing concentration of salts in the rhizosphere (Mahajan and
Tuteja 2005). This is a major concern in some parts of the world, particularly in arid and
semiarid areas, where evaporation greatly exceeds precipitation and salts dissolved in
the ground water reach and accumulate at the soil surface through capillary movement
(Kohler et al. 2010). Excessive soil salinization affects negatively the establishment,
growth and development of most plants and also of rhizosphere microbiota (Rietz and
Haynes 2003), leading to huge losses in plant productivity and diversity (Evelin et al.
2009). Some estimations suggest that salinization of arable land will result in 30% land
loss within next 25 years and up to 50% within next 40 years (Wang et al. 2003).
Salinity stress in plants depends on three main components: an initial osmotic
stress due to the reduction in the osmotic potential of the soil solution reduces the
amount of water available to the plant, causing physiological drought. This obliges the
plant to maintain lower internal osmotic potentials in order to prevent water movement
from roots into the soil (Feng et al. 2002; Jahromi et al. 2008). Secondly, the
accumulation of toxic ions, such as sodium and chloride, negatively affects cellular
metabolism (Munns et al. 2006). The toxic effects include disruption to the structure of
enzymes and other macromolecules, damage to cell organelles and plasma membrane,
disruption of photosynthesis, respiration and protein synthesis (Porcel et al. 2012).
Finally, salinity produces nutrient imbalance in the plant caused by decreased nutrient
uptake and/or transport to the shoot leading to ion deficiencies (Marschner 1995; Adiku
et al. 2001).
Salinity also induces an increase in the production of reactive oxygen species
(ROS), such as superoxide radicals (O2-), hydrogen peroxide (H2O2) and hydroxyl
radicals (OH•). These cytotoxic ROS can destroy normal metabolism through oxidative
damage of lipids, proteins, and nucleic acids when they are produced in excess (Miller
et al. 2010). Thus, efficient mechanisms are needed to prevent the possible oxidative
damage to cellular components.
As a consequence of all the above mentioned processes, salt stress affects plant
metabolism, including growth, photosynthesis, protein synthesis, and energy and lipid
metabolisms (Ramoliya et al. 2004). However, plants have evolved several biochemical
and molecular mechanisms to cope with the negative effects of salinity (Türkan and
Demiral 2009). In addition, besides the intrinsic adaptation mechanisms developed by
plants, in their natural environment they are associated to both saprophytic and
endophytic microorganisms, which can improve plant performance under stressful
conditions (Barea et al. 2005; Aroca and Ruiz-Lozano 2009).
Arbuscular mycorrhizal (AM) fungi are widespread microorganisms associated
symbiotically with the roots of 80% of terrestrial plants (van der Heijden et al. 1998;

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Smith and Read 2008). In the AM symbiosis, plants get nutrients and water resources
that are less available to the plant roots from the fungi, while the fungi receive carbon
compounds from the plant and find a niche to complete their life cycle (Koide and
Mosse 2004). At the same time, AM symbiosis enhances plant tolerance to different
abiotic stresses with osmotic components such as drought and salinity (Augé 2001;
Ruíz-Lozano 2003; Ruíz-Lozano et al. 2006; Jahromi et al. 2008). Although it is clear
that AM fungi mitigate growth reduction caused by salinity, the mechanism involved
remains unresolved (Ruiz-Lozano et al. 2012). Moreover, some studies reveal that
salinity affects directly the fungal development, reducing fungal mycelia formation and
host root colonization (Poss et al. 1985; Juniper and Abbott 2006; Giri et al. 2007;
Sheng et al. 2008). Contrary to those reports, a few other studies reported no reduction
or even increasing fungal development (Hartmond et al. 1987; Aliasgharzadeh et al.
2001; Yamato et al. 2008). This may be related to evolved mechanisms that allow
specific AM fungi to have a higher tolerance to salinity. In fact, mycorrhizal fungi have
been shown by several workers to occur naturally in saline environments (Juniper and
Abbott 1993) and Copeman et al. (1996) suggested that differences in fungal behaviour
and efficiency can be due to the origin of the AM fungus (Ruíz-Lozano and Azcón
2000). These results invite to look out for AM fungal species isolated in saline
environments and compare their salt-tolerance mechanisms with those of species living
in non-saline areas (Porcel et al. 2012).
The main problem to elucidate the mechanisms that allow specific AM fungi to
tolerate salinity is that salt tolerance is a multigenic and complex trait which involves
many physiological and biochemical mechanisms that vary between species (Mian et al.
2011). Thus, when examining putative salt tolerant AM fungi, several aspects involved
in the protection against damage caused by ROS, altered water content or protein
inactivation should be evaluated. In this regard, it is known that to overcome the
oxidative damage generated by stresses such as salinity, all living organisms have
developed antioxidant systems to efficiently scavenge ROS excess. Little is known
about the antioxidant responses in the AM fungi, but studies have demonstrated that
AM fungi possess ROS scavenging systems. These include genes encoding for
superoxide dismutases (SOD) (Lanfranco et al. 2005; González-Guerrero et al. 2010) or
glutaredoxins (GRXs) (Benabdellah et al. 2009), although their involvement in the
fungal responses to salinity have never been studied. On the other hand, aquaporins are
proteinaceous pores present in the membranes of all living organisms that facilitate the
transport of water and other small and neutral solutes (Forrest and Bhave 2007; Maurel
et al. 2008). An aquaporin gene has been described in the AM fungus G. intraradices
which may have a role in the transport of water from mycelium growing under
osmotically favourable conditions to salt-stressed mycelium (Aroca et al. 2009). Finally,
protection mechanisms to prevent protein inactivation by salinity can include the
activity of 14-3-3 proteins or that of chaperone-like proteins such as luminal binding
proteins (BiP). 14-3-3 proteins are binding proteins that regulate the activities of a wide

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array of targets via direct protein–protein interactions (Bridges and Moorhead 2004). In
plants, these proteins have a role in regulation of development and stress response
(Chung et al. 1999; Roberts 2003), including salinity (Wang et al. 2002; Xu and Shi
2006). A gene encoding for a 14-3-3 protein has been described in G. intraradices
(Porcel et al. 2006), but its involvement in the fungal response to salinity has not been
investigated so far. Luminal binding proteins (BiPs) are molecular chaperons present in
all kingdoms and their role in the endoplasmic reticulum is to transiently bind to
unfolded proteins to prevent intramolecular and intermolecular interactions that can
result in permanent misfolding or aggregation, with the subsequent loss of their function
(Gething and Sambrook 1992; Hendershot et al. 1996). A gene encoding for a BiP
protein has been described in G. intraradices but no information is available on its
possible biological function, although it was postulated that it could be similar to that of
animals or plants (Porcel et al. 2007).
In this study, a strain of G. intraradices isolated from Cabo de Gata Natural Park
(Almería, Spain), a region with serious problems of salinity and affected by
desertification, has been compared with a collection strain, used as a model fungus.
Bago et al. (1998b) highlighted monoxenic cultures as an appropriate tool for studying
the extraradical phase of AM symbiosis. In the present work we took advantage of the
in vitro monoxenic culture of AM fungi in order to study under increasing salinity
levels the differences in development and in expression of putative stress-responsive
genes of two strains of the AM fungus G. intraradices. In a parallel study, we
conducted an in vivo experiment with the same AM fungi and a host plant of agronomic
importance such as maize, in order to test their symbiotic efficiencies under increasing
salinity levels.

Materials and methods

Identification of the mycorrhizal strain isolated from Cabo de Gata Natural Park

AM fungal spores were separated from the soil samples by a wet sieving process
(Sieverding 1991). The morphological spore characteristics and their subcellular
structures were described from a specimen mounted in: polyvinyl alcohol-lactic acid-
glycerine (PVLG) (Koske and Tessier 1983); a mixture of PVLG and Melzer’s reagent
(Brundrett et al. 1994); a mixture of lactic acid to water at 1:1; Melzer’s reagent; and
water (Spain 1990). For identification of the AMF species, spores were then examined
using a compound microscope at up to 400-fold magnification as described by the
glomeromycotean classification of Oehl et al. (2011) recently published in the new
journal IMA Fungus. The species was clearly identified based on its spore morphology
as Glomus intraradices (Schenk and Smith 1982), which has been recently reassigned to
Rhizophagus intraradices (N.C. Schenck and G.S. Sm.) C. Walker & A. Schuessler

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2010. Spores presented a globose form, from 65 to 145 μm diameter, and light colour
between white and yellow (some old spores had a brownish colour). Up to four layers
were observed in some of the samples and some of them presented a hyphal constriction
at the base of the spore.
In addition to the morphological identification, a molecular identification was
also carried out. For that, spores isolated from the bait cultures were surface-sterilized
with chloramine T (2%) and streptomycin (0.02%) and crushed with a sterile disposable
micropestle in 40 μL milli-Q water (Ferrol et al. 2004). A two-step PCR was conducted
to amplify the AM fungal DNA from the spores. The first PCR step was performed with
the universal eukaryote primers NS1 and NS4 region of the small subunit ribosomal
gene and the second with the specific AM fungal primers AML1 and AML2 (Lee et al.
2008). The amplified DNA was purified using the Ilustra™ GFX™ PCR DNA and Gel
Band Purification Kit (GE Helthcare, UK). DNA fragments were sequenced on an
automated DNA sequencer (Perkin-Elmer ABI Prism 373). Sequence data were
compared to gene libraries (EMBL and GenBank) using BLAST program (Altschul et
al. 1990).
The BLAST analysis unambiguously placed Rhizophagus intraradices as the
closest relative of our G. intraradices CdG strain, with sequence accession number
FR750209 (Kruger et al. 2012) having a 99% identity.
The AM fungal strain has been incorporated to the collection of Zaidin
Experimental Station, Granada, Spain, under accession EEZ 195.

In vitro mycorrhizal cultures

The two Glomus intraradices strains used in this study were established in
monoxenic culture as described by St-Arnaud et al. (1996). For that, the clone DC2 of
carrot (Daucus carota L.) Ri-T DNA transformed roots were cultured in two-
compartment Petri dishes with the AM fungal strain DAOM 197198 of G. intraradices
Smith and Schenck [recently reassigned to G. irregulare by Stockinger et al. (2009) and
then as Rhizophagus irregularis (Błaszk., Wubet, Renker and Buscot) C. Walker and A.
Schüßler comb. nov.] or with the strain G. intraradices isolated from Cabo de Gata
(CdG) Natural Park in Almería (Southeast Spain; located at 36°44´41´´N,
02°07´26´´W).
The root compartment of each plate was filled with sterile minimal medium, as
described by Chabot et al. (1992) referred to as “M” throughout this report, to the top of
the division wall. The medium had been autoclaved for 20 minutes at 120ºC. The hyfal
compartment of each plate was filled in the same manner except that M medium used
did not contain any sucrose (referred to as “M-C” in this work). In addition, prior to the
sterilization of the M-C medium NaCl had been added to the medium to obtain 0, 75 or
150 mM concentrations of salt. The media were allowed to solidify at room

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temperature. There were six different treatments, with 50 replicate plates per treatment,
totalling 300 plates.
A piece of M agar (approximately 0.5 cm2) was cut from the root compartment
of each Petri dish and replaced with fungal inoculum, which consisted of a piece of agar
of the same size obtained from stock monoxenic cultures containing spores and hyphae
of the collection G. intraradices strain or the strain G. intraradices CdG. The
specimens, kept in continuous monoxenic cultures, were provided by the culture
collections of Zaidin Experimental Station, Granada, Spain. Three to four pieces of
transformed carrot roots (2.5 cm length each), grown in M medium, were placed on top
of the fungal inoculum in the root compartments. Two weeks after inoculation plates
were checked and if roots were crossing onto the distal compartments, they were
aseptically moved back to their proximal compartments. This check was subsequently
repeated once a week along the experiment. The plates were incubated in the dark at
24ºC for 2 months.

Parameters measured

Four, six and eight weeks after inoculation plates were examined under
dissecting microscope (Nikon, SMZ 1000, Japan) and AM fungal development was
assessed using the method described by Marsh (1971) and modified by Bago and Cano
(2005). A transparent 2 mm grid was used to determine the hyphal length, the number
of branched absorbing structures (BAS) (Bago et al. 1998a) and the number of spores in
three areas of 1 cm2 per distal compartment of each plate.

RNA extraction from fungal mycelium and synthesis of cDNA

After eight weeks, the mycelium from the distal compartment was isolated.
Citric acid monohydrate 10 mM at pH 6 was used to extract the mycelium from the M-
C medium. Then it was immersed in liquid nitrogen and stored at -80ºC until RNA was
extracted using the RNeasy plant mini kit (Qiagen, Valencia, CA, U.S.A.).
First single-strand cDNA was primed by random hexamers using 100–1,000 ng
of DNase-treated RNA. RNA samples were denatured at 65ºC for 5 min and then
reverse transcribed at 25ºC for 10 min and 42ºC for 50 min in a final volume of 20 μl
containing 10 μl of total RNA, 10 μM random primers (Invitrogen, Carlsbad, CA,
USA), 0.5 mM dNTPs, 10 U RNase inhibitor, 4 μl of 5x buffer, 2 μl 0.1 M DTT, and 1
μl of Superscript II Reverse Transcriptase (Invitrogen). The samples were precipitated
with 1 (v/v) isopropanol and suspended in 20 μl of water.

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Analysis of fungal gene expression

Gene expression analyses were carried out by quantitative reverse transcription


(qRT)-PCR using an iCycler iQ apparatus (BioRad, Hercules, CA, U.S.A.). The cDNA
samples were standardized to GintEF, For (5´-GCTATTTTGATCATTGCCGCC-3´)
and Rev (5´-TCATTAAAACGTTCTTCCGACC-3´) (González-Guerrero et al. 2005);
and Gint18S rRNA, For, (5′-TGTTAATAAAAATCGGTGCGTTGC-3′) and Rev, (5′-
AAAACGCAAATGATCAACCGGAC-3′) (González-Guerrero et al. 2005; Porcel et
al. 2006). The same reactions were performed with the specific primers for each of the
analyzed genes: GintSOD1 (For, 5´-GTACTATTACTTTCATTCAGGA-3´ and Rev,
5´-AGTTCATGACCACCTTTACCAA-3´) (González-Guerrero et al. 2005); Gint14-3-
3 (For, 5´-CGCAATCTCCTCTCAGTCGC-3´ and Rev, 5´-
GCAATAGCATCATCAAATGC-3´) (Porcel et al. 2006); GintBiP (For, 5´-
AGATGCTGGCGTAATTGCTGG-3´and Rev, 5´-TGGCGGCACCATATGCAACTG-
3´) (Porcel et al. 2007) and GintAQP1 (For, 5´-AGGACTCGGAGGTAGTGATGC-
3´and Rev, 5´-GCCGGATATATCACTCCAAAGC-3´) (Aroca et al. 2009).
Individual real-time RT-PCR reactions were assembled with oligonucleotide
primers (0.15 μM each), 10.5 μl of 2x iQSYBR Green Supermix (Bio-Rad; containing
100 mM KCl, 40 mM Tris–HCl pH 8.4, 0.4 Mm dNTPs, 50 U/μl iTaq DNA
polymerase, 6 mM MgCl2, 20 nM SYBR Green I, 20 nM fluorescein) plus 1 μl of a
1:10 dilution of each corresponding cDNA in a final volume of 21 μl. The PCR cycling
programme consisted of 4 min incubation at 95ºC, followed by 35 cycles of 45 s at
95ºC, 45 s at 60ºC and 45 s at 72ºC, where the fluorescence signal was measured.
Experiments were repeated three times, with the threshold cycle (CT) determined in
triplicate, using cDNAs originated from three RNAs extracted from three different
biological samples, each of them corresponded to a pool of three to five plates. The
relative levels of transcription were calculated by using the 2 -ΔΔCt method (Livak and
Schmittgen 2001). Negative controls without cDNA were used in all PCR reactions.

Statistical Analysis

Statistical analysis was performed using SPSS 19.0 statistical program (SPSS
Inc., Chicago, IL, USA) performing first a one-way ANOVA followed by the Tukey
test with P< 0.05 as the significance cut-off.

In vivo experiment

Soil and biological materials

The experiment consisted of a randomized complete block design with three


inoculation treatments: (1) noninoculated control plants, (2) plants inoculated with the

94
Chapter 3

AM fungal strain G. intraradices isolated from Cabo de Gata (CdG) and (3) plant
inoculated with the model AM fungus G. intraradices reproduced at collection of the
Zaidin Experimental Station. There were 30 replicates of each inoculation treatment,
totalling 90 pots (one plant per pot), so that ten pots of each microbial treatment were
grown under nonsaline conditions throughout the entire experiment, while ten pots per
treatment were subjected to 66 mM of NaCl and the remaining ten pots per treatment
were subjected to 100 mM of NaCl.
Loamy soil was collected from Granada province (Spain, 36º59’34’’N;
3º34’47’’W), sieved (5 mm), diluted with quartz-sand (<2 mm) (1:1, soil:sand, v/v) and
sterilized by steaming (100ºC for 1 h on 3 consecutive days). The original soil had a pH
of 8.2 [measured in water 1:5 (w/v)]; 1.5 % organic matter, nutrient concentrations (g
kg-1): N, 1.9; P, 1 (NaHCO3-extractable P); K, 6.9. The electrical conductivity of the
original soil was 0.5 dS m-1.
Three seeds of maize (Zea mays. L) were sown in pots containing 900 g of the
same soil/sand mixture as described above and thinned to one seedling per pot after
emergence.
Mycorrhizal inoculum was bulked in an open-pot culture of Zea mays L. and
consisted of soil, spores, mycelia and infected root fragments. The AM species used
were two strains of Glomus intraradices, the first one from our culture collection and
the second one isolated from Cabo de Gata (Almería, Spain). Appropriate amounts of
each inoculum containing about 700 infective propagules (according to the most
probable number test), were added to the corresponding pots at sowing time just below
maize seeds.
Uninoculated control plants received the same amount of autoclaved
mycorrhizal inocula together with a 10 ml aliquot of a filtrate (< 20 μm) of the AM
inocula in order to provide a general microbial population free of AM propagules.

Growth conditions

The experiment was carried out under glasshouse conditions with temperatures
ranging from 19 to 25ºC, 16/8 light/dark period, and a relative humidity of 70-80%. A
photosynthetic photon flux density of 800 μE m-2 s-1 was measured with a light meter
(LICOR, Lincoln, NE, USA, model LI-188B). Water was supplied daily to the entire
period of plant growth to avoid any drought effect. Plants were established for 45 days
prior to salinization to allow adequate plant growth and symbiotic establishment prior to
application of the salt stress. Three concentrations (0, 66, and 100 mM NaCl) of saline
solution were reached in the soil substrate by adding appropriate dilutions of a stock 2
M saline solution. The concentration of NaCl in the soil was increased gradually on
alternative days to avoid an osmotic shock. It took 8 days, to reach the desired 66 and
100 mM NaCl levels. The electrical conductivities in the soil were 0.25, 6.9 and 9.3 dS

95
Chapter 3

m-1 for the salt levels of 0, 66, and 100 mM NaCl, respectively. Plants were maintained
under these conditions for additional 30 days.

Parameters measured and statistical analysis

Symbiotic development

The percentage of mycorrhizal root infection in maize plants was estimated by


visual observation of fungal colonization after clearing washed roots in 10% KOH and
staining with 0.05% trypan blue in lactic acid (v/v), as described by Phillips and
Hayman (1970). The extent of mycorrhizal colonization was calculated according to the
gridline intersect method (Giovannetti and Mosse 1980).

Biomass production

At harvest (75 days after planting), the shoot and root system were separated and
the shoot dry weight (SDW) and root dry weight (RDW) were measured after drying in
a forced hot-air oven at 70ºC for two days.

Photosynthetic efficiency

The efficiency of photosystem II was measured with FluorPen FP100 (Photon


Systems Instruments, Brno, Czech Republic), which allows a non-invasive assessment
of plant photosynthetic performance by measuring chlorophyll a fluorescence. FluorPen
quantifies the quantum yield of photosystem II as the ratio between the actual
fluorescence yield in the light-adapted state (FV‘) and the maximum fluorescence yield
in the light-adapted state (FM‘), according to Oxborough and Baker (1997).
Measurements were taken in the third youngest leaf of ten different plants of each
treatment.

Stomatal conductance

Stomatal conductance was measured two hours after light turned on by using a
porometer system (Porometer AP4, Delta-T Devices Ltd, Cambridge, UK) following
the user manual instructions. Stomatal conductance measurements were taken in the
third youngest leaf from five different plants from each treatment.

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Chapter 3

Relative electrolyte leakage

The electrolyte leakage was measured as an index of salt injury to cellular


membranes (Verslues et al., 2006). It was calculated on the third leaf of each maize
plant from a leaf sample of 3 x 1.5 cm. The initial conductivity was measured with a
conductivity metre COND 510 (XS Instruments; OptoLab, Milan, Italy) after subjecting
the samples to incubation at 25ºC in 10ml de-ionized water overnight with continuous
shaking at 100 rpm. The samples were then autoclaved at 121ºC for 20 min. Final
conductivity was measured after the samples had cooled down to room temperature
(Verslues et al. 2006).

Results

Experiment in vitro

Fungal development under salt conditions

After four week of culture, the hyphal length produced by both AM fungal
strains was negatively affected by the presence of 75 and 150 mM NaCl in the medium
(Figure 1A). The effect of salinity decreasing hyphal length was similar for the two
fungal strains. At six weeks, only 150 mM NaCl decreased significantly the hyphal
length of both fungal strains as compared to the control treatment without salt (Figure
1B). Again, both AM fungal strains showed a similar hyphal length decrease. Finally,
after eight weeks of culture, Gi CdG did not show hyphal length reduction in response
to any of the salt levels applied to the medium (Figure 1C). In contrast, the collection G.
intraradices strain decreased the hyphal length after addition of 75 mM NaCl (26% of
decrease) and 150 mM NaCl (37% of decrease).
The number of spores produced by both fungal strains increased transiently at
four weeks after the application of 75 mM NaCl, while under 150 mM NaCl, it showed
similar levels than those of the no stress treatment (Figure 2A). At six weeks Gi CdG
showed no effect of salt on the number of spores produced, while the collection G.
intraradices strain increased significantly the number of spores produced after the
addition of 150 mM NaCl (Figure 2B). Finally, at eight weeks, both fungal strains
enhanced the number of spores produced after the addition of either 75 or 150 mM
NaCl, with no significant differences between both strains (Figure 2C).

97
Chapter 3

1500 Gi CdG A
1200
Gi collect

900
a a
600

300 b b b b
0
0 mM 75 mM 150 mM

1500 B
Hyphal lenght (cm)

1200 a ab
abc bc bc
900 c
600

300

0
0 mM 75 mM 150 mM

1500 a a a C
ab
1200
bc
900 c
600
300
0
0 mM 75 mM 150 mM

Figure 1. Total hyphal length (cm) formed in the hyphal compartment by two G. intraradices strains
grown in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars represents strain Gi CdG
and grey bars represent the collection G. intraradices strain. Measurements were done after 4 weeks (A),
6 weeks (B) or 8 weeks (C) of fungal growth in the medium. Means followed by different letters are
significantly different (P<0.05).

100 Gi CdG
A
80
Gi collect

60
40
20 ab a
b b b b
0
00mM
mM 75
75mM
mM 150
150 mM
mM

100
B
Number of spores

80
60 a
40 b
b b b
20
b
0
00 mM
mM 75
75 mM
mM 150
150 mM
mM

100 a C
a a a
80
60 b
40 b
20
0
00 mM
mM 75
75 mM
mM 150
150 mM
mM

Figure 2. Total number of spores formed in the hyphal compartment by two G. intraradices strains
grown in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars represents strain Gi
CdG and grey bars represent the collection G. intraradices strain. Measurements were done after 4 weeks
(A), 6 weeks (B) or 8 weeks (C) of fungal growth in the medium. Means followed by different letters are
significantly different (P<0.05).

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Chapter 3

After four weeks of culture, the number of BAS produced was negatively
affected by the highest salt level (150 mM) applied, with a similar decrease of this
parameter in both fungal strains (Figure 3A). At week 6, salinity influenced differently
the number of BAS produced by each fungal strain (Figure 3B). Thus, in Gi CdG, this
parameter was decreased by 150 mM NaCl, while the collection G. intraradices strain
did not show significant changes as a consequence of salt application. However, after
eight weeks of fungal culture no significant differences in BAS production were
observed among treatments (Figure 3C).
Gi CdG
90 Gi collect A

60 a a
ab
30 ab
b b
0
00 mM
mM 75
75 mM
mM 150
150 mM
mM

90
a ab B
ab a
Number of BAS

60
ab b
30

0
00 mM
mM 75
75 mM
mM 150
150 mM
mM

90 a C
a a
60 a a
a
30

0
00 mM
mM 75
75 mM
mM 150
150 mM
mM

Figure 3. Total number of BAS formed in the hyphal compartment by two G. intraradices strains grown
in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars represents strain Gi CdG and
grey bars represent the collection G. intraradices strain. Measurements were done after 4 weeks (A), 6
weeks (B) or 8 weeks (C) of fungal growth in the medium. Means followed by different letters are
significantly different (P<0.05).

Expression of chaperone-encoding genes

The most remarkable results were those related to the expression of the two
chaperone-encoding genes Gint14-3-3 and GintBIP (Figures 4 and 5). The expression of
both genes was considerably higher in Gi CdG than in the collection G. intraradices
strain, even in absence of NaCl in the growing medium (up regulation by 85 fold
Gint14-3-3 and by 96 fold GintBIP). The presence of salt in the medium further up
regulated the expression of these two genes in Gi CdG, mainly at 150 mM NaCl (up
regulation of Gint14-3-3 by 9 fold as compared to 0 mM NaCl or up regulation of
GintBIP by 13 fold as compared to 0 mM NaCl). In contrast, in the case of the
collection G. intraradices strain, only the application of 75 mM NaCl up regulated the

99
Chapter 3

expression of this gene, while the application of 150 mM NaCl down regulated or kept
unchanged the expression of Gint14-3-3 and GintBIP genes, respectively.

260 Gi CdG
a
Gi collect
210

160

110
Gint14-3-3 Relative expression

b
60
c

10

1,0
0,9
0,8
0,7
0,6
0,5 d
0,4
e
0,3
f
0,2
0,1
0,0
0 mM 75 mM 150 mM

Figure 4. Analysis of Gint14-3-3 gene expression by real time quantitative RT-PCR in two G.
intraradices strains grown in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars
represents strain Gi CdG and grey bars represent the collection G. intraradices strain. Means followed by
different letters are significantly different (P<0.05).

Gi CdG a

30,20 Gi collect

25,20

20,20

15,20 b
GintBiP Relative expression

10,20

5,20 c

0,20

0,10
0,09
0,08
0,07 d
0,06
0,05
0,04 e
0,03 e
0,02
0,01
0,00
0 mM 75 mM 150 mM

Figure 5. Analysis of GintBiP gene expression by real time quantitative RT-PCR in two G. intraradices
strains grown in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars represents strain
Gi CdG and grey bars represent the collection G. intraradices strain. Means followed by different letters
are significantly different (P<0.05).

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Chapter 3

Expression of a SOD-encoding gene

We analyzed the expression of a SOD encoding gene in the two G. intraradices


strains (Figure 6). The results showed that, in contrast to the previous genes, the
expression of this gene was higher in the collection G. intraradices strain than in Gi
CdG, even in absence of salt in the medium (up regulation by 75 fold). The addition of
75 mM of NaCl did not affect the expression of this gene in any of the two fungal
strains, while the application of 150 mM NaCl enhanced the expression of this gene
both in the collection G. intraradices strain (4 fold induction) and in Gi CdG (60 fold
induction). In spite of the strong induction of this gene in Gi CdG, the expression level
of this gene continued being higher in the collection G. intraradices strain.

0,93 Gi CdG
a
0,83 Gi collect
0,73
GintSOD1 Relative expression

0,63
0,53
0,43
0,33
b
0,23 b b
0,13
0,03

0,030

0,025

0,020

0,015

0,010

0,005 c c

0,000
0 mM 75 mM 150 mM

Figure 6. Analysis of GintSOD1 gene expression by real time quantitative RT-PCR in two G.
intraradices strains grown in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars
represents strain Gi CdG and grey bars represent the collection G. intraradices strain. Means followed by
different letters are significantly different (P<0.05).

Expression of an aquaporin-encoding gene

The expression of the GintAQP1 gene in Gi CdG and collection strains increased
alter the application of 150 and 75 mM NaCl, respectively. In the collection strain
however, addition of 150 mM NaCl to the culture medium caused no significant effect
on the expresión of this gene (Figure 7). At 75 mM Na Cl the expression of GintAQP1
was higher in the collection G. intraradices strain than in Gi CdG, while at 150 mM
NaCl it was just the opposite.

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Chapter 3

GintAQP1 Relative expression


0,040 Gi CdG ab
0,035
Gi collect
0,030
a
0,025
0,020
bc
0,015
0,010 c c
c
0,005
0,000
0 mM 75 mM 150 mM

Figure 7. Analysis of GintAQP1 gene expression by real time quantitative RT-PCR in two G.
intraradices strains grown in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars
represents strain Gi CdG and grey bars represent the collection G. intraradices strain. Means followed by
different letters are significantly different (P<0.05).

Experiment in vivo

Shoot and root dry weights (SDW and RDW)

Salt stress did not affect significantly SDW in non-AM plants, while both AM
treatments reduced SDW at 100 mM NaCl as compared to the non-salt stressed
treatment (Figure 8A). In any case, plants inoculated with Gi CdG exhibited the highest
SDW production at all salt levels studied. Indeed, the increases in SDW at 0 and 100
mM NaCl were, respectively, 26 and 17%, as compared to non-AM plants. Plants
inoculated with the collection G. intraradices strain exhibited similar SDW values than
non-AM plants at all salt levels.
The RDW showed no significant changes as a consequence of either salt levels
applied or the AM fungal strain inoculated (Figure 8B).

Symbiotic development

The percentage of mycorrhization was significantly higher in plants inoculated


with the Gi collection strain than in those inoculated with Gi CdG (Figure 9B). Under
the non-saline conditions plants inoculated with the collection G. intraradices strain had
67% of mycorrhizal root length. This was significantly lower than at 66 and 100 mM
NaCl, where the root infection reached 88% and 84%, respectively. Plants inoculated
with Gi CdG reached about 20% mycorrhizal root length, with no significant
differences among salt treatments (Figure 9B).

102
Chapter 3

Shoot dry weight (g.plant-1)


6 a A
5 ab
bcd cd cd bc
4 de cde
e
NM
3
Gi collect
2 Gi CdG

1
0
0 mM 66 mM 100 mM
Root dry weight (g.plant-1)

2,5 a B
a ab
2 ab ab ab ab
ab b
1,5

0,5

0
0 mM 66 mM 100 mM
-1
Figure 8. Shoot (A) and root (B) dry weights (g plant ) in maize plants. Black bars represent
noninoculated control plants (NM), grey bars represent plants inoculated with the collection G.
intraradices strain and white bars represent plants inoculated with the native strain Gi CdG. Plants were
subjected to 0, 66 or 100 mM NaCl. Columns with different letters are significantly different (P<0.05).

25
Electrolyte Leakage (%)

a
B
A
20 ab ab ab
ab ab
15 c c c

10

5
0
0 mM 66 mM 100 mM
(%)

100 a B
a
AM root colonization

80 b
60
%

40 c
c c
20
0
0 mM 66 mM 100 mM

Figure 9. Electrolyte leakage (A) and percentage of mycorrhizal root length (B) in maize plants. Black
bars represent noninoculated control plants (NM), grey bars represent plants inoculated with the
collection G. intraradices strain and white bars represent plants inoculated with the native strain Gi CdG.
Plants were subjected to 0, 66 or 100 mM NaCl. Columns with different letters are significantly different
(P<0.05).

103
Chapter 3

Relative electrolyte leakage

The applied salt stress did not significantly increase the relative electrolyte
leakage in maize plants from any treatment (Figure 9A). Under non saline conditions,
plants colonized by Gi CdG exhibited 25% less electrolyte leakage than non-AM plants,
while no significant effect was observed in plants inoculated with the collection G.
intraradices strain. At 66 mM NaCl, results were similar than under non saline
conditions. Finally, at 100 mM NaCl both AM fungi decreased electrolyte leakage as
compared to non-AM plants. In fact, plants inoculated with the collection G.
intraradices strain decreased this parameter by 22% and those inoculated with Gi CdG
did it by 36%.

Stomatal conductance

Under the non-saline conditions, there were no significant differences among


treatments in stomatal conductance (Figure 10A). The application of salt greatly
affected this parameter, which decreased in all treatments, even at 66 mM NaCl.
However, at 66 mM NaCl no significant differences in stomatal conductance were
found among treatments. In contrast, at 100 mM NaCl, Gi CdG exhibited 45% more
stomatal conductance than non-AM plants and 82% more than plants inoculated with
the collection G. intraradices strain.

Photosystem II efficiency

The efficiency of photosystem II was assessed by measuring chlorophyll a


fluorescence (Figure 10B). For all treatments, this parameter was only reduced by
salinity after the application of 100 mM NaCl. Under non-stressed conditions (0 mM
NaCl) and under moderate salinity (66 mM NaCl), the efficiency of photosystem II was
similar in AM and non-AM plants. In contrast, significant differences among treatments
were found at 100 mM of NaCl. In fact, non-AM plants reduced this parameter by 41%
as compared to 0 mM NaCl. In the case of AM treatments, the reduction was 21% and
19% for the collection G. intraradices strain and Gi CdG, respectively. At this salt level
both AM treatments exhibited significantly higher efficiency of photosystem II than
non-AM plants. Thus, AM plants showed 40% and 66% higher values of this parameter
than non AM plants after inoculation with the collection G. intraradices strain or with
Gi CdG, respectively.

104
Chapter 3

30,00
30,00 ab a
Stomatal conductance 25,00
25,00 a A
(mmolH2Om-2s-1)
20,00
20,00 b
bc
bc bc b
15,00
15,00 cd
cd
d
10,00
10,00 NM
Gi collect
5,00
5,00 Gi CdG
0,00
0,00
00 mM
mM 66
66 mM
mM 100 mM
100 mM
Photosynthetic efficiency

0,7 a
ab ab
ab B
0,6 b ab b
0,5 c
0,4 d
0,3
0,2
0,1
0
0 mM 66 mM 100 mM
-2 -1
Figure 10. Stomatal conductance (mmol H2O m s ) (A) and efficiency of photosystem II (B) in maize
plants. Black bars represent noninoculated control plants (NM), grey bars represent plants inoculated
with the collection G. intraradices strain and white bars represent plants inoculated with the native strain
Gi CdG. Plants were subjected to 0, 66 or 100 mM NaCl. Columns with different letters are significantly
different (P<0.05).

Discussion

The beneficial effects of different AM fungi on plant growth under saline


conditions have been demonstrated in various plant species (Ruíz-Lozano et al. 1996;
Feng et al. 2002; Yano-Melo et al. 2003; Cho et al. 2006; Zuccarini and Okurowska
2008; Wu et al. 2010). However, the symbiotic efficiency of AM fungi can vary
according to their origin and the growing conditions (Porcel et al. 2012). Indeed, AM
fungi can be found under extreme saline conditions, and they can be adapted to these
conditions (Wilde et al. 2009). Native AM fungi from areas affected by osmotic stresses
may potentially cope with salt stress in a more efficient way than other fungi (Ruiz-
Lozano and Azcón 2000; Querejeta et al. 2006). Estrada-Luna and Davies (2008)
observed that the association of Opuntia albicarpa with a mixture of fungi from
Sonoran Desert enhanced the nutrient concentrations in the plant more than ZAC-19 (an
experimental biofertilizer) and a G. intraradices obtained from a culture collection.
Cabo de Gata Natural Park is an arid region of south-east Spain which is subjected to
desertification and has important levels of soil salinity. In this work we isolated a G.
intraradices strain from Cabo de Gata Natural Park and we tested its symbiotic
efficiency with maize plants growing under salt stress. Results showed that Gi CdG
stimulated the growth of maize plants under two levels of salinity more than the
collection G. intraradices strain.

105
Chapter 3

Salt stress inhibits plant photosynthetic ability, which leads to a decrease in crop
production (Pitman and Läuchli 2002), but several publications report that AM fungi in
saline soils can decrease plant yield losses by increasing their photosynthetic capacity
(Reviewed by Evelin et al. 2009; Ruiz-Lozano et al. 2012). This agrees with results
obtained in this work with maize plants inoculated with Gi CdG. Moreover, plants
inoculated with the collection G. intraradices strain had higher percentage of root
colonization than those inoculated with Gi CdG, stressing that the symbiotic efficiency
of Gi CdG in terms of plant growth is higher than that of the collection G. intraradices.
The higher symbiotic efficiency of Gi CdG was also corroborated by the enhanced
efficiency of photosystem II and stomatal conductance and the lower electrolyte leakage
exhibited by maize plants under the different conditions assayed. This agrees with
previous reports that found higher photosynthetic efficiency in leaves of mycorrhizal
plants under saline conditions (Sheng et al. 2008; Zuccarini and Okurowska 2008),
higher stomatal conductance (Ruíz-Lozano et al. 1996; Jahromi et al. 2008; Sheng et al.
2008) and improved integrity and stability of the cellular membranes (Feng et al. 2002;
Garg and Manchanda 2008; Kaya et al. 2009). Stahl and Smith (1984) reported that
Agropyron smithii colonized with G. microcarpum collected from a desert had
increased stomatal opening under arid condition than that colonized with G.
microcarpum collected from a more mesic site. This is an example to show a specific
AM fungal strain being more adapted to specific environmental condition.
The presence of salts in the growth medium may induce changes in the length
and other morphological properties of the hyphae, thus affecting their symbiotic
efficiency and also their infective capacity. Indeed, some studies state that salt inhibits
spore germination or other fungal propagules, colonization of the plant roots and
sporulation of AM fungi (Juniper and Abbott 2006; Giri et al. 2007; Sheng et al. 2008;
Jahromi et al. 2008). In this study, the collection G. intraradices strain showed a
transient enhancement in the number of spores and BAS structures at 6 weeks after
growing, with no significant differences at 8 weeks. This could be regarded as a
symptom of stress perception in this fungal strain, because spores are a form of
resistance propagules that can survive under adverse conditions and BAS are thought to
be associated with the formation of spores (Bago et al. 1998a). In contrast, the hyphal
length was reduced in both fungal strains by salt application, but at 8 weeks after
growing this decrease was significantly higher in the collection G. intraradices than in
Gi CdG. As the mycelium is not a form of resistance propagule in AM fungi, a higher
hyphal development can be considered in terms of tolerance, being the AM fungus from
Cabo de Gata a more tolerant strain than the collection G. intraradices. Previous reports
have indicated that hyphal networks are a very important source for the rapid initiation
of root colonization (McGee et al. 1997; Smith and Read 2008). The maintenance of
AM fungi in ecosystems is dependent on the persistence of a potential inoculum in soils
(Brundrett 1991). Carvalho et al. (2004) found evidence for potential adaptation of
indigenous AM fungi to salt marsh conditions and for the ability of different propagules

106
Chapter 3

of these fungi to colonize new plants and spread the infection through the roots. Brito et
al. (2011) also showed that extraradical mycelium of native AM fungi can survive the
dry and hot summer in a typical Mediterranean region and initiate colonization of wheat
plants at the onset of the growing season; the same may occur with Gi CdG.
To determine the possible causes of the different behaviour and tolerance of both
AM fungal strains under saline conditions, we evaluated the effects of salinity on
several fungal genes potentially involved in the response to salinity. The induction of
genes encoding for chaperones, ROS scavengers, as well as, water channels is important
to re-establish cellular homeostasis and membranes stability during stresses (Xiong and
Zhu 2002; Bhatnagar-Mathur et al. 2008).
All the genes studied were up-regulated by increasing salinity in the fungus
isolated from Cabo de Gata, while the collection G. intraradices strain showed only an
up-regulation of the GintSOD1 gene. The overexpression of these genes under saline
conditions indicates that they have a role in the response of the fungus against osmotic
stress. Indeed, chaperone-like proteins, such as 14-3-3 and BiPs, have been
demonstrated to confer tolerance to a variety of stresses in plants, while in fungi the
literature is scarce. It has been proposed that BiP overexpression may prevent the cell
from sensing osmotic stress-induced variations in ER function by keeping ER basic
activities to a normal level under saline conditions (Valente et al. 2009). This is because
protein folding in the ER is facilitated by molecular chaperones, which prevent
nonproductive intermolecular interactions of folding intermediates and subsequent
misaggregation of proteins within the lumen of the ER (Hammond and Helenius 1995).
The AM fungal gene GintBIP, was studied in vitro by Porcel et al. (2007): they added
25% of PEG to the medium and the GintBIP gene expression increased by 41%. When
the gene was analyzed in vivo using maize, soybean and tobacco plants inoculated with
G. intraradices, the gene showed even higher expression. This was concomitant with
improved tolerance to drought (Porcel et al. 2007).
In a previous study, Porcel et al. (2006) found that the addition of PEG to the
medium increased Gint14-3-3 gene expression by 1200%. Expression of the gene was
also up-regulated in roots of mycorrhizal maize, lettuce and tobacco but not in soybean
where it did not show any change. It was proposed that Gint14-3-3 protein could
regulate the activity of plasma membrane H+-ATPases of either the fungus or the host
plant, to activate its pumping activity, which is essential to cope with osmotic stress
(Palmgren 1998). Indeed, the activity of plasma membrane H+-ATPase is highly
regulated by factors that affect the cell physiology, including stress conditions and
enhanced ATPase activity is crucial for the protective system that different organisms
have developed against external adverse influence (Palmgren 1998). Moreover, as 14-3-
3 proteins are found in association with key control enzymes of primary metabolism, its
overexpression could rapidly alter metabolic flux in response to signals such as salt
stress (Finnie et al. 1999) and they could also regulate the expression of stress-inducible

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genes by regulating the activity and or localization of transcription factors (Muslin and
Xing 2000).
Like other abiotic stresses, salinity also induces oxidative stress in plants
(Hajiboland and Joudmand 2009). In the field of the AM symbiosis, several studies
suggested that AM symbiosis helps plants to alleviate salt stress by enhancing the
activities of antioxidant enzymes (Alguacil et al. 2003; Zhong Qun et al. 2007; Garg
and Manchanda 2009; Talaat and Shawky 2011), but the response of the individual
enzymes varies with respect to the host plant and the fungal species involved in the
association. From the fungal side, González-Guerrero et al. (2010) described a
GintSOD1 gene encoding a functional protein that scavenges ROS. The up-regulation of
GintSOD1 transcripts in the fungal mycelia treated with paraquat and Cu indicated that
the gene product might be involved in the detoxification of the ROS induced by these
two external agents. Lanfranco et al. (2005) described and orthologous gene of
Gigaspora margarita, which may play a pivotal role in the relationship of the fungus
with its host plant, as it has been described in the ericoid mycorrhizal fungus
Oidiodendron maius (Abbà et al. 2009). As far as we know, this is the first study on the
effect of salinity on the expression of GintSOD1, showing that the gene is up-regulated
under saline conditions and providing evidence for a role of GintSOD1 in the fungal
response to the oxidative stress induced by salinity. In any case, the up-regulation of
this gene was lower in Gi CdG, suggesting that under salinity the accumulation of ROS
by this fungal strain could be lower than by the collection G. intraradices strain.
Salinity decreases the water potential of the medium, hampering the uptake of
water from the growing medium. Thus, the activity of aquaporins should be important
to living organisms in order to cope with the water deficit induced by salt stress.
Although it is well known that mycorrhizal mycelium transports water from the soil to
the roots, only three reports have studied mycorrhizal fungal aquaporins (Aroca et al.
2009; Dietz et al. 2011; Navarro-Ródenas et al. 2012). Dietz et al. (2011) reported that,
in the aquaporin gene family of Laccaria bicolor, three out of seven L. bicolor
membrane intrinsic proteins showed high water permeability and two of them were also
found to increase ammonia transport. Navarro-Ródenas et al. (2012) found high levels
of water conductivity of TcAQP1 that could be related to the adaptation of Terfezia
claveryi to semiarid areas because, as it was shown in a previous study, the mycelium of
this mycorrhizal fungus exhibited drought tolerance under in vitro conditions (Navarro-
Ródenas et al. 2012). However, only one study has been done so far on aquaporins from
an AM fungus (Aroca et al. 2009). Authors found some evidences supporting the idea
that fungal aquaporins could compensate the down regulation of host plant aquaporins
caused by osmotic stress. They also found that GintAQP1 expression was up regulated
in the osmotically non-stressed part of the mycelium when the other mycelium part was
stressed by NaCl. In the present study we found an up-regulation of GintAQP1 gene at
75 mM NaCl in the isolate from collection, but not in Gi CdG. In contrast, at the highest
salinity level (150 mM NaCl) the up regulation was found only in Gi CdG. Thus, Gi

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CdG has the ability to induce the expression of this aquaporin gene when the salt in the
medium reaches high levels. The biological significance of the up-regulation of
GintAQP1 gene remains to be elucidated since it was not possible to demonstrate
whether the respective aquaporin protein indeed transport water or other substrates
(Aroca et al. 2009).
In conclusion, results from this study show that the strain Gi CdG exhibited a
higher tolerance to salinity than the collection G. intraradices strain and grew and
developed better under saline conditions. The present study demonstrates that the Gi
CdG strain exhibits a better symbiotic efficiency in an already established symbiosis
under conditions of salt stress. These effects could be due to a fungal adaptation to the
saline environment where the fungus was isolated. The adaptation to salinity may be
related to the significant up-regulation of genes with chaperone activity or genes
encoding for aquaporins. The fungus from Cabo de Gata may reduce the production of
ROS, which in turns was evidenced by a lower induction of GintSOD1 gene.
The present study underlines the importance of salt adaptation in AM fungi.
Stress tolerance can only be gained through long-term exposure to chronic stress. Thus
AM fungi isolated from areas affected by salinity will be a powerful strategy to enhance
the tolerance of crops to saline stress conditions or in revegetation programs of
degraded areas affected by osmotic environmental constrains.

Acknowledgements

This work was financed by two research projects supported by Junta de


Andalucía (Spain). Projects P06-CVI-01876 and P11-CVI-7107. We thank Ascensión
Valderas and Jose Luis Manella for technical assistance in the in vitro experiment.

109
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References

Abbà S, Khouja HR, Martino E, Archer DB, Perotto S (2009) SOD1-targeted gene
disruption in the ericoid mycorrhizal fungus Oidiodendron maius reduces
conidiation and the capacity for mycorrhization. Mol Plant-Microbe Interact
22:1412-1421
Adiku SGK, Renger M, Wessolek G, Facklam M, Hecht-Bucholtz C (2001) Simulation
of the dry matter production and seed yield of common beans under varying soil
water and salinity conditions. Agric Water Manage 47:55-68
Alguacil MM, Hernández JA, Caravaca F, Portillo B, Roldán A (2003) Antioxidant
enzyme activities in shoots from three mycorrhizal shrub species afforested in a
degraded semi-arid soil. Physiol Plant 118:562-570
Aliasgharzadeh N, Rastin NS, Towfighi H, Alizadeh A (2001) Occurrence of arbuscular
mycorrhizal fungi in saline soils of the Tabriz Plain of Iran in relation to some
physical and chemical properties of soil. Mycorrhiza 11:119-122
Altschul SF, Gish W, Miller W, Myers EW, Lipton DJ (1990) Basic local alignment
search tool. J Mol Biol 215:403-410
Aroca R, Bago A, Sutka M, Paz JA, Cano C, Amodeo G, Ruíz-Lozano JM (2009)
Expression analysis of the first arbuscular mycorrhizal fungi aquaporin
described reveals concerted gene expression between salt-stressed and
nonstressed mycelium. Mol Plant-Microbe Interact 22:1169-1178
Aroca R, Ruiz-Lozano JM (2009) Induction of plant tolerance to semi-arid
environments by beneficial soil microorganisms. In: Lichtfouse E (ed) Climate
Change, Intercropping, Pest Control and Beneficial Microorganisms, sustainable
Agriculture Reviews 2. Springer, Dordrecht, The Netherlands, pp 121-135.
Augé RM (2001) Water relations, drought and vesicular-arbuscular mycorrhizal
symbiosis. Mycorrhiza 11:3-42
Bago B, Azcón-Aguilar C, Goulet A, Piché Y (1998a) Branched absorbing structures
(BAS): a feature of the extraradical mycelium of symbiotic arbuscular
mycorrhizal fungi. New Phytol 139:375-388
Bago B, Azcón-Aguilar C, Piché Y (1998b) Architecture and developmental dynamics
of the external mycelium of the arbuscular mycorrhizal fungus Glomus
intraradices grown under monoxenic conditions. Mycologia 90:52-62
Bago B, Cano C (2005) Breaking myths on arbuscular mycorrhizas in vitro biology. In
Vitro Culture of Mycorrhizas Vol 4 Soil Biology. pp. 111-138
Barea JM, Pozo MJ, Azcón R, Azcón-Aguilar C (2005) Microbial co-operation in the
rhizosphere. J Exp Bot 56:1761-1778
Benabdellah K, Merlos MA, Azcon-Aguilar C, Ferrol N (2009) GintGRX1, the first
characterized glomeromycotan glutaredoxin, is a multifunctional enzyme that
responds to oxidative stress. Fungal Genet Biol 46:94-103

110
Chapter 3

Bhatnagar-Mathur P, Vadez V, Sharma KK (2008) Transgenic approaches for abiotic


stress tolerance in plants: retrospect and prospects. Plant Cell 27:411-424
Bridges D, Moorhead GBG (2004) 14-3-3 proteins: a number of functions for a
numbered protein. Sci STKE 296:1-8
Brito I, de Carvalho M, Goss MJ (2011) Summer survival of arbuscular mycorrhiza
extraradical mycelium and the potential for its management through tillage
options in Mediterranean cropping systems. Soil Use Manage 27:350-356
Brundrett M (1991) Mycorrhizas in natural ecosystems. Adv Ecol Res 21:171-313
Brundrett M, Melville L, Peterson L (1994) Practical methods in mycorrhizal research.
Mycologue Publications, Ontario, Canada.
Carvalho LM, Correia PM, Martins-Louçao MA (2004) Arbuscular mycorrhizal fungal
propagules in a salt marsh. Mycorrhiza 14:165-170
Copeman RH, Martin CA, Stutz JC (1996) Tomato growth in response to salinity and
mycorrhizal fungi from saline or nonsaline soils. J Hortic Sci 31:341-344
Chabot S, Becard G, Piché Y (1992) Life-cycle of Glomus intraradix in root organ-
culture. Mycologia 84:315-321
Cho KH, Toler H, Lee J, Ownley B, Stutz JC, Moore JL, Auge RM (2006) Mycorrhizal
symbiosis and response of sorghum plants to combined drought and salinity
stresses. J Plant Physiol 163:517-528
Chung HJ, Sehnke PC, Ferl RJ (1999) The 14-3-3 proteins: cellular regulators of plant
metabolism. Trends Plant Sci 4:367-371
Dietz S, von Bülow J, Beitz E, Nehls U (2011) The aquaporin gene family of the
ectomycorrhizal fungus Laccaria bicolor: lessons for symbiotic functions. New
Phytol 190:927-940
Estrada-Luna AA, Davies FT (2008) Estado nutrimental y crecimiento de plantas
micropropagadas de nopal (Opuntia albicarpa Scheinvar cv. ‘Reyna’)
colonizadas con tres cepas seleccionadas de endomicorrizas. In: Montaño-Arias,
N. M, Camargo Ricalde, S. L, García-Sánchez, R, Monroy-Ata, A. (eds)
Micorrizas Arbusculares en Ecosistemas Áridos y Semiáridos. Grupo Mundi-
Prensa. México, pp 203-213
Evelin H, Kapoor R, Giri B (2009) Arbuscular mycorrhizal fungi in alleviation of salt
stress: a review. Ann Bot 104:1263-1280
Feng G, Zhang FS, Li XL, Tian CY, Tang C, Rengel Z (2002) Improved tolerance of
maize plants to salt stress by arbuscular mycorrhiza is related to higher
accumulation of soluble sugars in roots. Mycorrhiza 12:185-190
Ferrol N, Calvente R, Cano C, Barea JM, Azcón-Aguilar C (2004) Analyzing
arbuscular mycorrhizal fungal diversity in shrub-associated resource islands
from a desertification-threatened semiarid Mediterranean ecosystem. Appl Soil
Ecol 25:123-133
Finnie C, Borch J, Collinge DB (1999) 14-3-3 proteins: Eukaryotic regulatory proteins
with many functions. Plant Mol Biol 40:545-554

111
Chapter 3

Flowers TJ (2004) Improving crop salt tolerance. J Exp Bot 55:307-319


Forrest KL, Bhave M (2007) Major intrinsic proteins (MIPs) in plants: A complex gene
family with major impacts on plant phenotype. Funct Integr Genomic 7:263-289
Garg N, Manchanda G (2008) Effect of arbuscular mycorrhizal inoculation of salt-
induced nodule senescence in Cajanus cajan (pigeonpea). J Plant Growth Regul
27:115-124
Garg N, Manchanda G (2009) Role of Arbuscular Mycorrhizae in the Alleviation of
Ionic, Osmotic and Oxidative Stresses Induced by Salinity in Cajanus cajan (L.)
Millsp (pigeonpea). J Agron Crop Sci 195:110-123
Gething MJ, Sambrook J (1992) Protein folding in the cell. Nature 355:33-45
Giovannetti M, Mosse B (1980) Evaluation of techniques for measuring vesicular
arbuscular mycorrhizal infection in roots. New Phytol 84:489-500
Giri B, Kapoor R, Mukerji KG (2007) Improved tolerance of Acacia nilotica to salt
stress by arbuscular mycorrhiza, Glomus fasciculatum may be partly related to
elevated K/Na ratios in root and shoot tissues. Microb Ecol 54:753-760
González-Guerrero M, Azcón-Aguilar C, Mooney M, Valderas A, MacDiarmid CW,
Eide DJ, Ferrol N (2005) Characterization of a Glomus intraradices gene
encoding a putative Zn transporter of the cation diffusion facilitator family.
Fungal Genet Biol 42:130-140
González-Guerrero M, Oger E, Benabdellah K, Azcón-Aguilar C, Lanfranco L, Ferrol
N (2010) Characterization of a CuZn superoxide dismutase gene in the
arbuscular mycorrhizal fungus Glomus intraradices. Curr Genet 56:265-274
Hajiboland R, Joudmand A (2009) The K/Na replacement and function of antioxidant
defence system in sugar beet (Beta vulgaris L.) cultivars. Acta Agr Scand B-S P
59:246-259
Hammond C, Helenius A (1995) Quality control in the secretory pathway. Curr Opin
Cell Biol 7:523-529
Hartmond U, Schaesberg NV, Graham JH, Syversten JP (1987) Salinity and flooding
stress effects on mycorrhizal and non-mycorrhizal citrus rootstock seedlings.
Plant Soil 104:37-43
Hendershot L, Wei J, Gaut J, Melnick J, Aviel S, Argon Y (1996) Inhibition of
immunoglobulin folding and secretion by dominant negative BiP ATPase
mutants. Proc Natl Acad Sci USA 93:5269-5274
Jahromi F, Aroca R, Porcel R, Ruiz-Lozano JM (2008) Influence of salinity on the In
vitro development of Glomus intraradices and on the In vivo physiological and
molecular responses of mycorrhizal lettuce plants. Microb Ecol 55:45-53
Juniper S, Abbott L (2006) Soil salinity delays germination and limits growth of hyphae
from propagules of arbuscular mycorrhizal fungi. Mycorrhiza 16:371-379
Juniper S, Abbott LK (1993) Vesicular-arbuscular mycorrhizas and soil salinity.
Mycorrhiza 4:45-57

112
Chapter 3

Kaya C, Ashraf M, Sonmez O, Aydemir S, Tuna AL, Cullu MA (2009) The influence
of arbuscular mycorrhizal colonization on key growth parameters and fruit yield
of pepper plants grown at high salinity. Scientia Hortic 121:1-6
Kohler J, Caravaca F, Roldán A (2010) An AM fungus and a PGPR intensify the
adverse effects of salinity on the stability of rhizosphere soil aggregates of
Lactuca sativa. Soil Biol Biochem 42:429-434
Koide RT, Mosse B (2004) A history of research on arbuscular mycorrhiza. Mycorrhiza
14:145-163
Koske RE, Tessier B (1983) A convenient, permanent slide mounting medium. Myc
Soc America Newsletter 34:59
Kruger M, Kruger C, Walker C, Stockinger H, Schussler A (2012) Phylogenetic
reference data for systematics and phylotaxonomy of arbuscular mycorrhizal
fungi from phylum to species level. New Phytol 193:970-984
Lanfranco L, Novero M, Bonfante P (2005) The mycorrhizal fungus Gigaspora
margarita possesses a CuZn superoxide dismutase that is up-regulated during
symbiosis with legume hosts. Plant Physiol 137:1319-1330
Lee J, Lee S, Young JPW (2008) Improved PCR primers for the detection and
identification of arbuscular mycorrhizal fungi. FEMS Microbiol Ecol 65:339-
349
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-
time quantitative PCR and the 2-ΔΔCt method. Methods 25:402-408
Mahajan S, Tuteja N (2005) Cold, salinity and drought stresses: an overview. Arch
Biochem Biophys 444:139-158
Marschner H (1995) Mineral nutrition of higher plants second edition. Academic Press,
London, UK
Marsh B (1971) Measurement of length in random arrangement of lines. J Appl Ecol
8:265
Maurel C, Verdoucq L, Luu D-T, Santoni V (2008) Plant aquaporins: Membrane
channels with multiple integrated functions. Annu Rev Plant Biol 59:595-624
McGee PA, Pattinson GS, Heath RA, Newman CA, Allen SJ (1997) Survival of
propagules of arbuscular mycorrhizal fungi in soils in eastern Australia used to
grow cotton. New Phytol 135:773-780
Mian AA, Senadheera P, Maathuis FJM (2011) Improving Crop Salt Tolerance: Anion
and Cation Transporters as Genetic Engineering Targets. Plant Stress 5:64-72
Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2010) Reactive oxygen species
homeostasis and signalling during drought and salinity stresses. Plant Cell
Environ 33:453-467
Munns R, James RA, Läuchli A (2006) Approaches to increasing the salt tolerance of
wheat and other cereals. J Exp Bot 57:1025-1043
Muslin AJ, Xing H (2000) 14-3-3 proteins: Regulation of subcellular localization by
molecular interference. Cell Signal 12:703-709

113
Chapter 3

Navarro-Ródenas A, Ruiz-Lozano JM, Kaldenhoff R, Morte A (2012) The Aquaporin


TcAQP1 of the Desert Truffle Terfezia claveryi Is a Membrane Pore for Water
and CO2 Transport. Mol Plant-Microbe Interact 25:259-266
Oehl F, Sieverding E, Palenzuela J, Ineichen K, Alves da silva G (2011) Advances in
Glomeromycota taxonomy and classification. IMA Fungus 2:191-199
Oxborough K, Baker NR (1997) Resolving chlorophyll a fluorescence images of
photosynthetic efficiency into photochemical and non-photochemical
components - calculation of qP and Fv '/Fm ' without measuring Fo '. Photosynth
Res 54:135-142
Palmgren MG (1998) Proton gradients and plant growth: role of the plasma membrane
H+-ATPase. Adv Bot Res 28:1–70
Phillips JM, Hayman DS (1970) Improved procedure of clearing roots and staining
parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of
infection. Trans Br Mycol Soc 55:159-161
Pitman M, Läuchli A (2002) Global impact of salinity and agricultural ecosystems. In:
Läuchli A, Lüttge, U. (ed) Salinity: environment–plants–molecules. Springer
Verlag, Netherlands, pp 3-20
Porcel R, Aroca R, Cano C, Bago A, Ruíz-Lozano JM (2006) Identification of a gene
from the arbuscular mycorrhizal fungus Glomus intraradices encoding for a 14-
3-3 protein that is up-regulated by drought stress during the AM symbiosis.
Microb Ecol 52:575-582
Porcel R, Aroca R, Cano C, Bago A, Ruíz-Lozano JM (2007) A gene from the
arbuscular mycorrhizal fungus Glomus intraradices encoding a binding protein
is up-regulated by drought stress in some mycorrhizal plants. Environ Exp Bot
60:251-256
Porcel R, Aroca R, Ruiz-Lozano JM (2012) Salinity stress alleviation using arbuscular
mycorrhizal fungi. A review. Agron Sustain Develop 32: 181-200
Poss JA, Pond E, Menge JA, Jarrell WM (1985) Effect of salinity on mycorrhizal onion
and tomato in soil with and without additional phosphate. Plant Soil 88:307-319
Querejeta JI, Allen MF, Caravaca F, Roldán A (2006) Differential modulation of host
plant δ13C and δ18O by native and nonnative arbuscular mycorrhizal fungi in a
semiarid environment. New Phytol 169:379-387
Ramoliya PJ, Patel HM, Pandey AN (2004) Effect of salinization of soil on growth and
macro- and micro-nutrient accumulation in seedlings of Salvadora persica
(Salvadoraceae). Forest Ecology Management 202:181-193
Rietz DN, Haynes RJ (2003) Effects of irrigation-induced salinity and sodicity on soil
microbial activity. Soil Biol Biochem 35:845-854
Roberts MR (2003) 14-3-3 proteins find new partners in plant cell signaling. Trends
Plant Sci 8:218-223
Ruiz-Lozano JM (2003) Arbuscular mycorrhizal symbiosis and alleviation of osmotic
stress. New perspectives for molecular studies. Mycorrhiza 13:309-317

114
Chapter 3

Ruiz-Lozano JM, Azcón R (2000) Symbiotic efficiency and infectivity of an


autochthonous arbuscular mycorrhizal Glomus sp. from saline soils and Glomus
deserticola under salinity. Mycorrhiza 10:137-143
Ruiz-Lozano JM, Azcón R, Gómez M (1996) Alleviation of salt stress by arbuscular-
mycorrhizal Glomus species in Lactuca sativa plants. Physiol Plant 98:767-772
Ruiz-Lozano JM, Porcel R, Aroca R (2006) Does the enhanced tolerance of arbuscular
mycorrhizal plants to water deficit involve modulation of drought-induced plant
genes? New Phytol 171:693-698
Ruiz-Lozano JM, Porcel R, Azcón R, Aroca R (2012) Regulation by arbuscular
mycorrhizae of the integrated physiological response to salinity in plants. New
challenges in physiological and molecular studies. J Exp Bot 63: 4033-4044
Schenk NC, Smith GS (1982) Additional new and unreported species of mycorrhizal
fungi (Endogonaceae) from Florida. Mycologia 74:77-92
Sheng M, Tang M, Chen H, Yang B, Zhang F, Huang Y (2008) Influence of arbuscular
mycorrhizae on photosynthesis and water status of maize plants under salt stress.
Mycorrhiza 18:287-296
Sieverding E (1991) Vesicular-arbuscular mycorrhiza management in tropical
agrosystems. Technical Cooperation (GTZ). Eschborn. Friedland, Bremer,
Rossdorf, TZ-Verlagsgesellschaft, Germany.
Smith SE, Read DJ (2008) Mycorrhizal Symbiosis, 3rd Ed. Elsevier, Academic Press,
New York
Spain JL (1990) Arguments for diagnoses based on unaltered wall structures.
Mycotaxon 38:71-76
St-Arnaud M, Hamel C, Vimard B, Caron M, Fortin JA (1996) Enhanced hyphal growth
and spore production of the arbuscular mycorrhizal fungus Glomus intraradices
in an in vitro system in the absence of host roots. Mycol Res 100:328-332
Stahl PD, Smith WK (1984) Effects of different geographic isolates of Glomus on water
relations of Agropyron smithii. Mycologia 76:261-267
Schuessler A, Walker C (2010) The Glomeromycota: a species list with new families
and new genera 1-58. electronic publication. Available at: www.amf-
phylogeny.com
Stockinger H, Walker C, Schüßler A (2009) ‘Glomus intraradices DAOM197198’, a
model fungus in arbuscular mycorrhiza research, is not Glomus intraradices.
New Phytol 183:11761187
Talaat NB, Shawky BT (2011) Influence of arbuscular mycorrhizae on yield, nutrients,
organic solutes, and antioxidant enzymes of two wheat cultivars under salt
stress. J Plant Nutr Soil Sci 174:283-291
Tester M, Davenport R (2003) Na+ tolerance and Na+ transport in higher plants. Ann
Bot 91:503-527
Türkan I, Demiral T (2009) Recent developments in understanding salinity tolerance.
Environ Exp Bot 67:2-9

115
Chapter 3

Valente MAS, Faria JAQA, Soares-Ramos JRL, Reis PAB, Pinheiro GL, Piovesan ND,
Morais AT, Menezes CC, Cano MAO, Fietto LG, Loureiro ME, Aragão FJL,
Fontes EPB (2009) The ER luminal binding protein (BiP) mediates an increase
in drought tolerance in soybean and delays drought-induced leaf senescence in
soybean and tobacco. J Exp Bot 60:533-546
van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R,
Boller T, Wiemken A, Sanders IR (1998) Mycorrhizal fungal diversity
determines plant biodiversity, ecosystem variability and productivity. Nature
396:69-72
Verslues PE, Agarwal M, Katiyar-Agarwal S, Zhu J, Zhu JK (2006) Methods and
concepts in quantifying resistance to drought, salt and freezing, abiotic stresses
that affect plant water status. Plant J 45:523-539
Wang WX, Vinocur B, Altman A (2003) Plant responses to drought, salinity and
extreme temperatures: towards genetic engineering for stress tolerance. Planta
218:1-14
Wang YH, Garvin DF, Kochian LV (2002) Rapid induction of regulatory and
transporter genes in response to phosphorus, potassium, and iron deficiencies in
tomato roots: evidence for cross-talk and root/rhizosphere-mediated signals.
Plant Physiol 130:1361-1370
Wilde P, Manal A, Stodden M, Sieverding E, Hildebrandt U, Bothe H (2009)
Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt
marshes. Environ Microbiol 11:1548-1561
Wu QS, Zou YN, He XH (2010) Contributions of arbuscular mycorrhizal fungi to
growth, photosynthesis, root morphology and ionic balance of citrus seedlings
under salt stress. Acta Physiol Plant 32:297-304
Xiong L, Zhu JK (2002) Molecular and genetic aspects of plant responses to osmotic
stress. Plant, Cell Environ 25:131-139
Xu WF, Shi WM (2006) Expression profiling of the 14-3-3 gene family in response to
salt stress and potassium and iron deficiencies in young tomato (Solanum
lycopersicum) roots: analysis by real-time RT-PCR. Ann of Bot 98:965-974
Yamato M, Ikeda S, Iwase K (2008) Community of arbuscular mycorrhizal fungi in
coastal vegetation on Okinawa Island and effect of the isolated fungi on growth
of sorghum under salt-treated conditions. Mycorrhiza 18:241–249
Yano-Melo AM, Saggin OJ, Costa-Maia L (2003) Tolerance of mycorrhized banana
(Musa sp. cv. Pacovan) plantlets to saline stress. Agric, Ecosyst Environ 95:343-
348
Zhong Qun H, Chao Xing H, Zhibin Z, Zhirong Z, Huai Song W (2007) Changes in
antioxidative enzymes and cell membrane osmosis in tomato colonized by
arbuscular mycorrhizae under NaCl stress. Colloids Surf B 59:128-133
Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6:66-71

116
Chapter 3

Zuccarini P, Okurowska P (2008) Effects of mycorrhizal colonization and fertilization


on growth and photosynthesis of sweet basil under salt stress. J Plant Nutr
31:497-513

117
CAPÍTULO 4
CHAPTER 4

Arbuscular mycorrhizal fungi native from a Mediterranean saline area


enhance maize plants tolerance to salinity through improved ion
homeostasis

Submitted to Annals of Botany by Estrada B, Aroca R, Maathuis F.J.M,


Barea J.M and Ruiz-Lozano J.M.
Capítulo 4

Hongos micorrícico arbusculares nativos de áreas salinas


Mediterráneas aumentan la tolerancia de plantas de maíz mediante la
mejora de la homeostasis iónica

Resumen

La salinidad del suelo restringe seriamente el crecimiento y la productividad vegetal. El


maíz es una planta sensible a la sal, donde el Na+ representa el ión principal que causa
toxicidad, ya que puede competir con el K+ por sitios de unión en la membrana
plasmática. La inoculación con micorrizas arbusculares (MA) puede aliviar el estrés
salino en las plantas hospedadoras a través de varios mecanismos. Estos pueden incluir
la selección de iones durante la absorción de los nutrientes del suelo por parte de los
hongos o durante la transferencia a la planta huésped. Se ha propuesto que los
beneficios de las MA podrían verse aumentados usando aislados nativos de MA,
fisiológica y genéticamente adaptados a las condiciones de estrés de su entorno. En este
estudio se investigó si hongos nativos MA aislados del Parque Natural de Cabo de Gata
(CdG) (una zona con graves problemas de salinidad y afectada por la desertificación)
pueden ayudar a las plantas de maíz a superar los efectos negativos del estrés salino
mejor que plantas inoculadas con hongos MA no nativos o que plantas no micorrizadas .
Los resultados mostraron que las plantas de maíz inoculadas con dos de los tres hongos
nativos MA tuvieron el mayor desarrollo de parte aérea en todos los niveles de
salinidad, mientras que la biomasa de las plantas inoculadas con el aislado MA de
colección fue similar a las plantas no micorrizadas. Las plantas inoculadas con los tres
hongos nativos MA mostraron un aumento significativo de K+ y una reducción en la
acumulación de Na+ en comparación con las plantas no micorrizadas, coincidente con
una mayor relación K+/Na+ en sus tejidos. Estos efectos se correlacionaron con la
regulación de la expresión de los genes ZmAKT2, ZmSOS1 y ZmSKOR en las raíces de
las plantas de maíz colonizadas por hongos MA nativos, lo que contribuye a la
homeostasis de K+ y Na+.

Palabras clave: adaptación, homeostasis iónica, hongos micorrízicos arbusculares


nativos, salinidad, tolerancia

121
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Arbuscular mycorrhizal fungi native from a Mediterranean saline area


enhance maize plants tolerance to salinity through improved ion
homeostasis

Beatriz Estrada1, Ricardo Aroca1, Frans J.M. Maathuis2, José Miguel Barea1 and
Juan Manuel Ruiz-Lozano1*

1
Departamento de Microbiología del Suelo y Sistemas Simbióticos. Estación
Experimental del Zaidín (CSIC). Profesor Albareda nº 1, 18008 Granada, Spain.
2
Department of Biology Area 9, University of York, York YO10 5DD, UK

* Corresponding author: Dr. Juan Manuel Ruiz-Lozano.


e-mail: juanmanuel.ruiz@eez.csic.es
Telph. + 34 958 181600
Fax: + 34 958 129600

Running title: Improved ion homeostasis in maize plants by salt-adapted AMF

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Abstract

Soil salinity seriously restricts plant growth and productivity. Maize is a salt-sensitive
plant, where Na+ represents the major ion causing toxicity because it can compete with
K+ for binding sites at the plasma membrane. Inoculation with arbuscular mycorrhizal
fungi (AMF) can alleviate salt stress in several host plants through several mechanisms.
These may include ion selection during the fungal uptake of nutrients from the soil or
during transfer to the host plant. It has been proposed that AM benefits could be
enhanced when native AMF isolates, physiologically and genetically adapted to the
stress conditions of their environment, are used. In this study we investigated whether
native AMF isolated from Cabo de Gata Natural Park (CdG) (an area with serious
problems of salinity and affected by desertification) can help maize plants to overcome
the negative effects of salinity stress better than non-AM plants or plants inoculated
with non-native AMF. Indeed, results showed that maize plants inoculated with two out
the three native AMF had the highest shoot dry biomass at all salinity levels, while
biomass in plants inoculated with the collection AM strain was similar to the non-
mycorrhizal plants. Plants inoculated with the three native AMF showed significant
increase of K+ and reduced Na+ accumulation as compared to non-mycorrhizal plants,
concomitantly with higher K+/Na+ ratios in their tissues. These effects correlated with
regulation of ZmAKT2, ZmSOS1 and ZmSKOR genes expression in the roots of maize
plants colonized by native AMF, contributing to K+ and Na+ homeostasis.

Key words: adaptation, ion homeostasis, native arbuscular mycorrhizal fungi, salinity,
tolerance

Introduction

Salinity is a major and increasing problem which restricts plant growth and
productivity. More than 800 million hectares of land throughout the world are salt
affected (including both saline and sodic soils) (FAO, 2005). This is over 6% of the
total land area of the world. High amounts of salts in soils are responsible for yield
reduction in one third of the global arable land (Lambers, 2003). This is particularly the
case in regions with high rates of evaporation, like arid and semiarid areas (Hammer et
al., 2011). Most crops are glycophytic and tolerate salinity to a threshold level. Above
this level, yield decreases (Khan et al., 2006), since excess of salt inhibits
photosynthetic ability and induces physiological drought in plants (Pitman and Läuchli,
2002). Maize (Zea mays L.) is classified as a salt-sensitive plant (Maas and Hoffman,
1977). Although maize is originally from Mesoamerica, nowadays it is the third most

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important cereal crop and ranks first in countries with developing economies (Mejía,
2003).
Most glycophytes tolerate salinity by restricting the uptake of Na+ and Cl- while
maintaining uptake of macronutrients such as K+ or N (Teakle and Tyerman, 2010).
Although Cl- is considered an essential micronutrient for higher plants involved in the
regulation of important cellular functions such as enzyme activity, maintenance of
membrane potentials, and as a co-factor in photosynthesis and pH gradients (White and
Broadley, 2001), it can be toxic to plants at high concentrations (Xu et al., 2000).
However for maize, it has been shown that Na+ (and not Cl-) represents the major ion
causing toxicity related to salinity (Fortmeier and Schubert, 1995) because it can
compete with K+ for binding sites at the plasma membrane. The K+ ion is essential for
protein synthesis, activation of many enzymes and photosynthesis and it plays a central
role in osmotic adjustment, turgor maintenance, and in the control of stomata opening
(Maathuis and Amtmann, 1999). It has been shown that chloroplast function is impaired
when K+ is displaced by Na+, leading to uncontrolled water losses (Slabu et al., 2009).
Furthermore, adequate K+ is very important to maintain cytosolic ion homeostasis in
Na+-stressed plants (Zhu, 2003), a function which is disrupted by excessive Na+ entry
(Demidchik and Maathuis, 2007). Accumulation of Na+ and impairment of K+ nutrition
is a major characteristic of salt stressed plants, the mechanisms of which are only
partially understood. However, salt stress often causes reduction in plant tissue K+
content, and the K+/Na+ ratio is considered a useful parameter to assess salt tolerance
(Maathuis and Amtmann, 1999; Chen et al., 2007). Another important response of
glycophytes to salinity stress, associated with osmoregulation adjustment, is the
accumulation of osmotically active organic solutes such as proline and glycine-betaine
(Munns, 2005). Proline maintains the osmotic balance and protects enzymes in presence
of high cytoplasmic electrolyte concentrations (Greenway and Munns, 1980; Hajlaoui et
al., 2010). However, the significance of proline accumulation in osmotic adjustment is
still debated and varies according to the species (Lutts et al., 1996; Rodriguez et al.,
1997).
Plants can overcome salinity effects by interacting with several beneficial soil
microorganisms. Soil microbiota, such as arbuscular mycorrhiza fungi (AMF) live
symbiotically associated with the roots of 80% of terrestrial plants (Smith and Read,
2008) and are able to increase plant growth and crop productivity under different
environmental stresses (Barea et al., 2012). Several studies have shown that inoculation
with AMF can alleviate salt stress (Sannazzaro et al., 2006; Jahromi et al., 2008;
Estrada et al., 2012). Improved salt tolerance following mycorrhizal colonization may
be the result of a more efficient nutrient uptake (Cantrell and Linderman, 2001), ion
balance (Giri et al., 2007), protection of enzyme activities (Rabie and Almadini, 2005),
increase in photosynthesis ability (Sheng et al., 2008) and facilitation of water uptake in
plants (Aroca et al., 2007).

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Mycorrhizal colonization has also been shown to enhance K+ absorption under


saline conditions while preventing Na+ translocation to shoot tissues (Giri et al., 2007;
Sharifi et al., 2007; Talaat and Shawky, 2011). Thus, mycorrhizal plants grown under
saline conditions often have a higher K+:Na+ ratio (Rabie and Almadini, 2005;
Sannazzaro et al., 2006), and a lower shoot Na+ concentration (Al-Karaki and Hammad,
2001) than nonmycorrhizal plants, preventing the disruption of various enzymatic
processes and inhibition of protein synthesis. Mycorrhizal fungi may also act as a first
barrier for ion selection during the fungal uptake of nutrients from the soil or during
transfer to the plant host. It has been indicated that AMF can selectively take up
elements such as K+ and Ca2+, which act as osmotic equivalents while they avoid uptake
of toxic Na+ (Hammer et al., 2011; Evelin et al., 2012). This suggests that AMF induce
a buffering effect on the uptake of Na+ when the content of Na+ is within the
permissible limit (Evelin et al., 2009; Hammer et al., 2011). Indeed, analyzing the
regulation by AMF of plant genes involved in ion homeostasis has been encouraged in a
recent review on physiological and molecular perspectives in studies of salt stress
alleviation by AMF (Ruiz-Lozano et al., 2012). In this sense, the plasma membrane
localised Na+:H+ antiporter SOS1 has been shown to fulfil two important roles in plants,
restriction of net Na+ uptake by roots and control of xylem loading for long-distance
transport of Na+ (Shi et al., 2002). Transport of K+ to the shoot depends on xylem
delivery, a process largely controlled by SKOR (Gaymard et al., 1998) and on phloem
K+ recycling (Maathuis, 2007). The molecular mechanism for the latter is unclear but
likely to involve the phloem expressed K+ channel AKT2 (Marten et al., 1999). Thus,
these three genes were studied in this work.
AMF can be found under severe saline conditions in nature, both in saline
inlands and coasts (Aliasgharzadeh et al., 2001; Yamato et al., 2008) and in salt
marshes (Carvalho et al., 2004; Wilde et al., 2009). Moreover, the use of AMF adapted
to salinity could be a critical issue for success in recovering saline areas either in natural
environments or in agricultural lands affected by salinity. Several studies, describing
inoculation strategies used in re-vegetation of degraded ecosystems, showed a higher
benefit of native AMF, which appear to be physiologically and genetically adapted to
the stress conditions of the target environment, than non-native isolates (Ferrol et al.,
2004; Oliveira et al., 2005; Querejeta et al., 2006). This can be extrapolated to salt-
stressed soil, thus the use of salinity-adapted AMF ecotypes should be rewarding.
The objectives of this work were: (i) to investigate whether native AMF isolated
from a saline environment (Cabo de Gata Natural Park, Almería, Spain, an area with
serious problems of salinity and affected by desertification) can help maize plants to
overcome the negative effects of salinity stress better than non-AM plants or plants
inoculated with non-native AMF. (ii) As the molecular mechanisms involved in the
better performance of AM plants under salinity stress are almost completely unknown,
and there is little information on the effects of the AM symbiosis on plant ion

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transporters, we also analyzed the regulation by these AMF of key plant ion transporters
expected to be affected by salinity.

Materials and methods

Identification of the mycorrhizal strains isolated from Cabo de Gata Natural Park

AM fungal spores were separated from the soil samples by a wet sieving process
(Sieverding, 1991). The morphological spore characteristics and their subcellular
structures were described from a specimen mounted in: polyvinyl alcohol-lactic acid-
glycerine (PVLG) (Koske and Tessier, 1983); a mixture of PVLG and Melzer’s reagent
(Brundrett et al., 1994); a mixture of lactic acid to water at 1:1; Melzer’s reagent; and
water (Spain, 1990). For identification of the AMF species, spores were then examined
using a compound microscope at up to 400-fold magnification as described for
glomeromycotean classification by Oehl et al. (2011). The species were identified based
on its spore morphology as a Rhizophagus intraradices (Schenk and Smith, 1982),
Claroideoglomus etunicatum (Becker and Gerdemann, 1977) and Septoglomus
constrictum (Trappe, 1977).
In addition to the morphological identification, a molecular identification was
also carried out. For that, spores isolated from the bait cultures of each fungal strain
were surface-sterilized with chloramine T (2%) and streptomycin (0.02%) and crushed
with a sterile disposable micropestle in 40 μL milli-Q water (Ferrol et al., 2004). A two-
step PCR was conducted to amplify the AM fungal DNA from the spores. The first PCR
step was performed with the universal eukaryote primers NS1 and NS4 region of the
small subunit ribosomal gene and the second with the specific AM fungal primers
AML1 and AML2 (Lee et al., 2008). The amplified DNA was purified using the
Ilustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Helthcare, UK). DNA
fragments were sequenced on an automated DNA sequencer (Perkin-Elmer ABI Prism
373). Sequence data were compared to gene libraries (EMBL and GenBank) using
BLAST program (Altschul et al., 1990).
The BLAST analysis unambiguously placed Rhizophagus intraradices as the
closest relative of our Rhizophagus intraradices CdG strain, with sequence accession
number FR750209 (Krüger et al., 2012) having a 99% identity. Septoglomus
constrictum was the closest relative to our Se. constrictum CdG strain, with sequence
accession number FR750212 (Krüger et al., 2012) having a 99% identity. Finally,
Claroideoglomus etunicatum was the closest relative of our Cl. etunicatum CdG strain,
with sequence accession number FR750216.1 (Krüger et al., 2012) having also a 99%
identity. The AM fungal strains have been incorporated to the collection of Zaidin
Experimental Station, Granada, Spain, under accession numbers EEZ 195, EEZ 196 and
EEZ 163, respectively.

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Experimental design

The experiment consisted of a randomized complete block design with five inoculation
treatments: (1) non-mycorrhizal control plants, (2) plants inoculated with the model AM
fungus Rh. intraradices (Ri collect), reproduced at collection of the Zaidin
Experimental Station, (3) plants inoculated with the AM fungal strain Rh. intraradices
isolated from Cabo de Gata Natural Park (Ri CdG), (4) plants inoculated with the AM
fungal strain Se. constrictum isolated from CdG (Sc CdG) and (5) plants inoculated with
the AM fungal strain Cl. etunicatum isolated from CdG (Ce CdG). There were 30
replicates of each inoculation treatment, totalling 150 pots (one plant per pot), so that
ten of each microbial treatment were grown under nonsaline conditions throughout the
entire experiment, while ten pots per treatment were subjected to 66 mM of NaCl and
the remaining ten pots per treatment were subjected to 100 mM of NaCl.

Soil and biological materials

Loamy soil was collected from Granada province (Spain, 36º59’34’’N; 3º34’47’’W),
sieved (5 mm), diluted with quartz-sand (<2 mm) (1:1, soil:sand, v/v) and sterilized by
steaming (100ºC for 1 h on 3 consecutive days). The original soil had a pH of 8.2
[measured in water 1:5 (w/v)]; 1.5 % organic matter, nutrient concentrations (g kg-1): N,
1.9; P, 1 (NaHCO3-extractable P); K, 6.9. The electrical conductivity of the original soil
was 0.5 dS m-1.
Three seeds of maize (Zea mays. L) were sown in pots containing 900 g of the
same soil/sand mixture as described above and thinned to one seedling per pot after
emergence.

Inoculation treatments

Mycorrhizal inoculum was bulked in an open-pot culture of Zea mays L. and consisted
of soil, spores, mycelia and infected root fragments. The AM species used were three
strains isolated from Cabo de Gata Natural Park (Almería, Spain): Rhizophagus
intraradices (previously named Glomus intraradices), Septoglomus constrictum and
Claroideoglomus etunicatum. A Rhizophagus intraradices strain from our culture
collection was also used. Appropriate amounts of each inoculum containing about 700
infective propagules (according to the most probable number test), were added to the
corresponding pots at sowing time just below maize seeds. Non-mycorrhizal control
plants received the same amount of autoclaved mycorrhizal inocula together with a 10
ml aliquot of a filtrate (< 20 μm) of the AM inocula in order to provide a general
microbial population free of AM propagules.

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Growth Conditions

The experiment was carried out under glasshouse conditions with temperatures ranging
from 19 to 25ºC, 16/8 light/dark period, and a relative humidity of 50-60%. A
photosynthetic photon flux density of 800 μE m-2 s-1 was measured with a light meter
(LICOR, Lincoln, NE, USA, model LI-188B). Water was supplied daily to the entire
period of plant growth to avoid any drought effect. Plants were established for 45 days
prior to salinization to allow adequate plant growth and symbiotic establishment. Three
concentrations (0, 66, and 100 mM NaCl) of saline solution were reached in the soil
substrate by adding appropriate dilutions of a stock 2 M saline solution. The
concentration of NaCl in the soil was increased gradually on alternative days to avoid an
osmotic shock. It took 8 days, to reach the desired 66 and 100 mM NaCl levels. Plants
were maintained under these conditions for an additional 30 days.

Parameters measured and statistical analysis

Symbiotic development

The percentage of mycorrhizal root infection in maize plants was estimated by visual
observation of fungal colonization after clearing washed roots in 10% KOH and
staining with 0.05% trypan blue in lactic acid (v/v), as described Phillips and Hayman
(1970). The extent of mycorrhizal colonization was calculated according to the gridline
intersect method (Giovannetti and Mosse, 1980).

Biomass production

At harvest (75 days after planting), the shoot and root system were separated and the
shoot dry weight (SDW) and root dry weight (RDW) was measured after drying in a
forced hot-air oven at 70ºC for two days.

Proline content
Free proline was extracted from 0.5 g of fresh leaves and roots (Bligh and Dyer, 1959).
The methanolic phase was used for quantification of proline content. Proline was
estimated by spectrophotometric analysis at 530 nm of the ninhydrin reaction according
to Bates et al. (1973).

Determination of Mineral Nutrients

Na+ and K+ ions were extracted from 0.05 g of ground leaf and root dry material with 10
ml of deionized water. This extract was diluted, and for analysis, an ICP plasma
analyzer (IRIS Intrepid II XDL, Thermo Electron Corporation) was used. Extractions

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were made from five different plants of each treatment. Mineral analyses were carried
out by the Analytical Service of the Centro de Edafología y Biología Aplicada del
Segura, CSIC, Murcia, Spain.
Chloride anions were determined from an aqueous extraction from 0.4 g of fresh
vegetal material with 10 ml of deionized water following the method of Cataldo et al.
(1975). The quantification of Cl- in roots and leaves was made from five different plants
of each treatment as described by Diatloff and Rengel (2001).

Gene expression analysis

RNA extraction and synthesis of cDNA

RNA was extracted using the RNeasy plant mini kit (Qiagen, Valencia, CA, U.S.A.)
from maize roots samples stored at -80ºC. Single-strand cDNA was primed by random
hexamers using 100-1,000 ng of DNase-treated RNA. RNA samples were denatured at
65ºC for 5 min and then reverse transcribed at 25ºC for 10 min and 42ºC for 50 min in a
final volume of 20 μl containing 10 μl of total RNA, 10 μM random primers
(Invitrogen, Carlsbad, CA, USA), 0.5 mM dNTPs, 10 U RNase inhibitor, 4 μl of 5x
buffer, 2 μl 0.1 M DTT, and 1 μl of Superscript II Reverse Transcriptase (Invitrogen).
The samples were precipitated with 1 (v/v) isopropanol and suspended in 20 μl of water.

Quantitative PCR

Gene expression analyses were carried out by quantitative reverse transcription (qRT)-
PCR using an iCycler iQ apparatus (BioRad, Hercules, CA, U.S.A.). The cDNA
samples were standardized to four reference genes: alpha tubulin (gi:450292),
elongation factor 1-alpha (EF1-α) (gi:2282583), polyubiquitin (gi:248338) and
glyceraldehyde phosphate dehydrogenase (GADPH) (gi:22237). The same reactions
were performed with specific primers designed for each of the analyzed genes:
ZmAKT2, For (5´-CCTCAAGCATCAGGTCGAGA-3´) and Rev (5´-
CTCTGTAATCTTCCTGGACG-3´), ZmSKOR, For (5´-
TCAGATCCAAGATGTCCCAG-3´) and Rev (5´-TTCGTATCCTCTTAACGCAG-3´)
ZmSOS1, For (5´-GCTTGTCACATACTTCACAG-3´) and Rev (5´-
ACTTGTCCACTTCACTACAC-3´). Individual real-time RT-PCR reactions were
assembled with oligonucleotide primers (0.15 μM each), 10.5 μl of 2x iQSYBR Green
Supermix (Bio-Rad; containing 100 mM KCl, 40 mM Tris-HCl pH 8.4, 0.4 Mm dNTPs,
50 U/μl iTaq DNA polymerase, 6 mM MgCl2, 20 nM SYBR Green I, 20 nM
fluorescein) plus 1 μl of a 1:10 dilution of each corresponding cDNA in a final volume
of 21 μl. Experiments were repeated three times, with the threshold cycle (CT)
determined in triplicate, using cDNAs that originated from three RNAs extracted from
three different biological samples.

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The relative levels of transcript were calculated using the Normalization Factor
(NF) based on the expression levels of the three best-performing housekeeping genes, in
our case polyubiquitin, GADPH and EF1-α. NF was measured using a Visual Basic
application for excel (GeNorm) that calculates the gene stability as described by
Vandesompele et al. (2002). The calculation was done for each cDNA used in the Q-
PCR quantification. Expression levels were transformed from Cq values using the PCR
efficiencies (Ramakers et al., 2003).

Statistical Analysis

Statistical analysis was performed using SPSS 19.0 statistical program (SPSS Inc.,
Chicago, IL, USA) performing first a one-way ANOVA followed by the Tukey test
with P< 0.05 as the significance cut-off. Two independent statistical analyses were
carried out: the first to analyze data from the different AMF treatments within each
saline level and the second one to analyze data from each fungal species at increasing
salinity.

Results

Symbiotic development

Increasing salinity application enhanced the percentage of AM root colonization in all


cases, except in plants colonized by Ri CdG, which exhibited similar colonization rates
at all salinity levels (Fig. 1). Within each salinity level, the highest rate of AM root
colonization was achieved in plants inoculated with Ri collect (up to 88%). High levels
of root colonization were also found in plants inoculated with Ce CdG and Sc CdG (Fig.
1). In contrast, the lowest root colonization was always found in plants colonized by Ri
CdG (about 20%).

Plant biomass production

The increase of salt application affected negatively the shoot biomass production in all
treatments; although in the case of non-mycorrhizal plants the decrease was not
significant (Fig. 2A). In all AM treatments, the decrease was more evident at the highest
salt level applied (100 mM NaCl). In contrast, the root biomass production only
decreased with salinity in the non-mycorrhizal plants (Fig. 2B). When results were
analyzed for the three salt treatments it was clear that Ri CdG and Ce CdG, both
enhanced maize shoot biomass as compared to the non-mycorrhizal plants, while Ri
collect and Sc CdG did not (Fig. 2A).

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AM root colonization (%)


D
100 a W
E X J X a
80 b b J b NM
a a Ri collect
c Ri CdG
60 K Sc CdG
G b G G Ce CdG
40
c c d
20
0
0 mM 66 mM 100 mM

Fig. 1. Percentage of mycorrhizal root length in maize plants. Grey bars represent plants inoculated with
the collection Rhizophagus intraradices strain (Ri collect); white bars, plants inoculated with the native
Rh. intraradices CdG strain (Ri CdG); lined bars, plants inoculated with the native Septoglomus
claroideum CdG strain (Sc CdG) and dotted bars, plants inoculated with the native Claroideoglomus
etunicatum CdG strain (Ce CdG). Plants were subjected to 0, 66 or 100 mM NaCl. Different letters
indicate significant differences (p < 0.05) among fungal treatments at each salt level (a, b, c, d) or among
salt levels for each AMF treatment: Ri collect (D, E, F), Ri CdG (G, H, I), Sc CdG (J, K, L) or Ce CdG
(W, X, Y).

G W W
6
Shoot dry weight (g.plant -1)

a GH a X A
a H
5 A D J A a a
DE J A a
b b b b
4 b b b E K
c
c
3
NM
2 Ri collect
Ri CdG
1 Sc CdG
Ce CdG
0
0 mM 66 mM 100 mM
Root dry weight (g.plant -1)

3 W B
W G W
2.5 A G a
D a a
a D ab J B D G a B
ab J ab J
2
b ab b ab ab
b b
1.5
1

0.5

0
0 mM 66 mM 100 mM

Fig. 2. Shoot (A) and root (B) dry weights (g plant-1) in maize plants. Black bars represent non-
mycorrhizal control plants (NM); grey bars, plants inoculated with the collection Rhizophagus
intraradices strain (Ri collect); white bars, plants inoculated with the native Rh. intraradices CdG strain
(Ri CdG); lined bars, plants inoculated with the native Septoglomus claroideum CdG strain (Sc CdG) and
dotted bars, plants inoculated with the native Claroideoglomus etunicatum CdG strain (Ce CdG). Plants
were subjected to 0, 66 or 100 mM NaCl. Different letters indicate significant differences (p < 0.05)
among fungal treatments at each salt level (a, b, c, d) or among salt levels for each AMF treatment: NM
plants (A, B, C), Ri collect (D, E, F), Ri CdG (G, H, I), Sc CdG (J, K, L) or Ce CdG (W, X, Y).

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Accumulation of proline

The accumulation of proline was more pronounced in root than in shoot tissues (Fig.
3A,B) and it increased in the roots with increasing salinity in the growth medium,
except for plants inoculated with Sc CdG, where the differences were not significant
(Fig. 3B). The highest accumulation of proline occurred in the roots of non-mycorrhizal
plants at 100 mM NaCl. It also increased with salinity in roots of plants inoculated with
Ri CdG and Ce CdG. In shoots, no significant differences were found either as a
consequence of increasing salinity or by the AM fungus inoculated.

W
0.6
A a A
0.5 A G
a D a D a
A a J
0.4 G W ab J W
a D J G a
0.3 a a
a
a ab ab
b NM
Proline content (μmol.g-1 DW)

0.2 Ri collect
Ri CdG
0.1 Sc CdG
Ce CdG
0
0 mM 66 mM 100 mM

A
2 a B
D G
1.5 AB ab ab W
a DE J W bc
ab H J
1 B ab ab c
J b
a E
I a X
0.5 a
a a

0
0 mM 66 mM 100 mM

Fig. 3. Shoot (A) and root (B) proline accumulation in maize plants. See legend for Fig. 2.

Accumulation of mineral ions in shoots and roots and K+/Na+ ratios

Potassium

The increase of salinity in the growing medium decreased the accumulation of K+ in the
root tissues in all treatments, except in plants inoculated with Ri collect, which had
similar K+ levels at 0 mM NaCl and at 100 mM NaCl (Fig. 4B). In contrast, in shoot
tissues, maize accumulated more K+ at increasing salinity levels (Fig. 4A). This was
especially evident in plants inoculated with Sc CdG. At 100 mM NaCl, all the
mycorrhizal treatments accumulated more K+ in roots than the non-mycorrhizal plants
(Fig. 4B). At this salt level no significant differences in K+ accumulation were observed

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in roots between AM and non-AM treatments. In shoots, at all salinity levels, the non-
mycorrhizal plants and the plants inoculated with Ri collect exhibited always a lower K+
accumulation than plants inoculated with either of the three native AM fungal strains
(Ri CdG, Sc CdG or Ce CdG) (Fig. 4A).
J
J a
4
G a W G
W A
3.5 K ab b
H a X A DE
b b
3 a A D
a c c c
2.5 B E c
2 b b NM
Ri collect
1.5
Ri CdG
1 Sc CdG
0.5 Ce CdG
K+ content (%)

0
0 mM 66 mM 100 mM

2 B
G J
D
a aW
1.5 A a a DE H
H K X K X
a a a
B E ab ab a B a a
1 b ab b

0.5

0
0 mM 66 mM 100 mM

Fig. 4. Shoot (A) and root (B) potassium concentration in maize plants. See legend for Fig. 2.

Sodium

The accumulation of Na+ in maize plants increased considerably both in shoot and in
root tissues when the plants were cultivated under salinity (Fig. 5 A,B). When data were
analyzed within each salt level, it was observed that at 0 mM NaCl the three native
AMF enhanced the accumulation of Na+ in root tissues as compared to the non-
mycorrhizal plants or those inoculated with Ri collect (Fig. 5B). However, at 66 mM
NaCl and 100 mM NaCl no significant differences in Na accumulation in roots were
observed among treatments, except that plants inoculated with Ce CdG, reduced
significantly this parameter at 100 mM NaCl. In the shoot tissues, it was observed that
at 0 mM NaCl the levels of Na+ were very low in all treatments (Fig. 5A). The
accumulation of Na+ was enhanced at 66 and 100 mM NaCl for all treatments, with
non-mycorrhizal plants exhibiting the highest Na+ accumulation and mycorrhizal plants
the lowest, especially those inoculated with Ce CdG (Fig. 5A).

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2.5 A
A
a
2
D J
B
1.5 b G b
a bc
E W NM
H K Ri collect
1 b c
b b X Ri CdG
b
Na+ content (%)

0.5 C F I L Y Sc CdG
a a a a a Ce CdG
0
0 mM 66 mM 100 mM

A J
3.5
B G a DG a B
3 E a ab
ab a K W W
ab ab b
2.5 b
2
H L X
1.5
ab b a
1 C F
0.5 c c
0
0 mM 66 mM 100 mM

Fig. 5. Shoot (A) and root (B) sodium concentration in maize plants. See legend for Fig. 2.

A
16 G J
B H K a D A
ab W
14 E bc b
a ab a X c
12 bc
I L c
Y
10 C F b a
c NM
8 d cd
Ri collect
6
Ri CdG
Cl- content (mmol.g-1 DW)

4 Sc CdG
2 Ce CdG
0
0 mM 66 mM 100 mM

J
J A
30 D a
B G a W a D G W B
J a a a
25 a a a a
a
20 H X
C E
ab ab
15 b ab
10
5
0
0 mM 66 mM 100 mM

Fig. 6. Shoot (A) and root (B) chloride concentration in maize plants. See legend for Fig. 2.

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Chloride

The accumulation of Cl- increased in both shoot and root tissues with increasing salinity
in the growth medium (Fig. 6 A,B). This was more evident in the shoot tissues (Fig.
6A). When data were analyzed within each salt level, it was observed that in roots no
remarkable differences in Cl- accumulation were found among treatments. In shoots, at
0 mM NaCl, plants inoculated with the three native AMF (Ri CdG, Sc CdG and Ce
CdG) accumulated more Cl- than non-mycorrhizal plants or those inoculated with the Ri
collect fungus (Fig. 6A). In contrast, when salt was applied to the growth medium, non-
mycorrhizal plants always exhibited the highest Cl- accumulation and no important
differences in Cl- accumulation were observed among fungal treatments. At both saline
levels, plants inoculated with Ce CdG showed the lowest accumulation of Cl-.

J
D
400 a
a G A
350 ab
300 A
bc NM
250 W
c Ri collect
200 Ri CdG
Sc CdG
150
Ce CdG
100

15
X
a
10 K
H X
b
E bc K a
5
B
cd E H
B b
d cd bc
d
K+/Na+ ratio

0
0 mM 66 mM 100 mM

A D
4 a a B
3.5
3
2.5
G J
2
b b W
1.5 b
K X H X
1 B E H B E ab K
ab a a
0.5 c bc bc c ab bc
0
0 mM 66 mM 100 mM

Fig. 7. Shoot (A) and root (B) K+/Na+ ratio in maize plants. See legend for Fig. 2.

136
Chapter 4

K+/Na+ ratios

The K+/Na+ ratio was negatively affected by salinity in both shoots and roots (Fig.
7A,B). However, the effect was more evident in shoot tissues, where the differences
between the non-saline treatment and either of the two saline treatments were of two
orders of magnitude (Fig. 7A). In roots, the K+/Na+ ratio at 0 mM NaCl was lower in
plants inoculated with either of the three native AMF as compared to non-mycorrhizal
plants or plants inoculated with Ri collect (Fig. 7B). In contrast, when salt was applied
non-mycorrhizal plants showed the lowest K+/Na+ ratio, especially if compared to roots
of plants inoculated with Ce CdG. In the shoots, at both saline levels, the lowest K+/Na+
ratios were also found in non-mycorrhizal plants and in plants colonized with the Ri
collect strain (Fig. 7A). The three native AMF (Ri CdG, Sc CdG and Ce CdG) showed
significantly enhanced K+/Na+ ratios in shoots as compared to the non-mycorrhizal
plants, especially those colonized by Ce CdG.

Expression of genes encoding for ion transporters

Ion analyses suggest that AMF affect tissue K+ and Na+. We therefore tested whether
membrane transporters involved in shoot K+ and Na+ deposition were affected at the
transcript level by AMF colonization.
The expression of the ZmAKT2 gene was differently affected by increasing
salinity in the different fungal treatments (Fig. 8A). In fact, in roots of non-mycorrhizal
plants or plants colonized by the Ri collect strain, it decreased its expression at 66 and
100 mM NaCl as compared to 0 mM NaCl. In contrast, the expression of this gene
increased steadily with increasing salinity in roots of plants colonized by Sc CdG and
Ce CdG. When data were analyzed within each salt level, it was observed that in the
absence of salt in the growth medium the expression of ZmAKT2 was notably higher in
roots of non-mycorrhizal plants and plants colonized by the Ri collect strain than in
roots of plants colonized by either of the three native AM fungal strains. At 66 mM
NaCl, few differences among treatments were observed. Finally, at the highest salt level
(100 mM NaCl), the expression of this gene increased notably in roots of plants
colonized by Sc CdG and Ce CdG, as compared to the other treatments.
The expression of the ZmSOS1 gene was negatively affected by the highest
salinity level in non-mycorrhizal plants or plants inoculated with Ri collect and Ri CdG
(Fig. 8B). In contrast, the application of 100 mM NaCl enhanced considerably the
expression of the ZmSOS1 gene in roots of plants colonized by Ce CdG as compared to
66 mM NaCl or to the non-saline level. When data were analyzed within each salt level,
it was observed that in the absence of salinity plants inoculated with Sc CdG or with Ce
CdG had a significantly lower expression of ZmSOS1 than the non-mycorrhizal plants.
No significant differences among treatments were observed at 66 mM NaCl, while at

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Chapter 4

the highest salt level (100 mM NaCl) plants inoculated with Ce CdG exhibited the
highest expression of this gene in their roots.
The expression of the ZmSKOR gene in roots was little affected by the
increasing salinity in non-mycorrhizal plants or in plants inoculated with the Ri collect
and Ri CdG strains (Fig. 8C). In contrast, in plants colonized by Sc CdG and Ce CdG,
the expression of this gene increased steadily with increasing salinity. When data were
analyzed within each salt level, it was observed that in the absence of salinity plants
inoculated with Sc CdG or with Ce CdG had a significantly lower expression of
ZmSKOR than all other treatments. This lower expression was maintained at 66 mM
NaCl in roots of plants colonized by Ce CdG, but again, at the highest salt level (100
mM NaCl) plants inoculated with Ce CdG and also plants inoculated with Sc CdG
exhibited the highest expression of this gene.
W
ZmAKT2 relative expression

2 a
J A
a
1.5
A D
a a
1 B
K NM
G b Ri collect
a X G
0.5 b L X C H E b Ri CdG
ab Sc CdG
b b b b c Ce CdG
0
0 mM 66 mM 100 mM
ZmSOS1 relative expression

1 W
A a B
D
0.8 a D
ab G a G
J
ab X AB a a X B E JK
0.6
a a bc bc b
K b
0.4 c H
c
0.2

0
0 mM 66 mM 100 mM
ZmSKOR relative expression

2 C
J
1.5
a W
G A D
1 a a a K a
A DE A H
a
a a b E b
X H X
0.5 L b
b b b
b
0
0 mM 66 mM 100 mM

Fig. 8. Analysis of ZmAKT2 (A), ZmSOS1 (B) and ZmSKOR (C) genes expression by real time quantitative
RT-PCR in roots of maize plants from the different treatments. See legend for Fig. 2.

138
Chapter 4

Discussion

Previous studies have demonstrated that maize plants inoculated with AMF grow better
than non-mycorrhizal plants under salt stress conditions (Feng et al., 2002; Sheng et al.,
2008; Sheng et al., 2011). However these experiments were based on the inoculation of
a sole AM fungus, Glomus mosseae. Although mycorrhizal symbiosis is usually
considered nonspecific, Klironomos (2003) found that there was great variation in
growth performance with the same fungal isolate. Therefore, AM colonization would
depend on the compatibility of both AMF and host plants. In the present work, we
assessed the performance of three AM fungal species isolated from a saline area and
compared their effectiveness with a G. intraradices (= Rhizophagus intraradices) strain
belonging to the Zaidin Experimental Station collection. Each species exhibited
different mycorrhizal development and symbiotic efficiency under the salt levels
assayed.
The results of the present study showed that colonization rates varied among
fungal species. Ri collect had the higher rate of root colonization, followed by Ce CdG,
Sc CdG and Ri CdG. While most studies reported a decrease in mycorrhizal
colonization at increasing salinity levels (Sharifi et al., 2007; Evelin et al., 2012), our
study reveals a significant increase in root colonization in three of the AMF strains, Ri
collect, Sc CdG and Ce CdG, while Ri CdG had the same colonization rate at all salinity
levels. Yamato et al. (2008) also found that colonization rates were not reduced in any
of the AMF present in coastal vegetation on Okinawa Island. Similar results were
reported by Wu et al. (2010) in citrus colonized by Paraglomus occultum isolated from
a saline habitat. These results may be explained in terms of salt-tolerance of the AMF
isolates: they can maintain or even increase colonization capacity under saline
conditions. On the other hand, Ri collect has been previously described to have a very
high rate of colonization (Graham et al., 1996; Ruiz-Lozano et al., 2001), thus it seems
not surprising that it maintained or even increased the colonization rate. Nevertheless,
under saline conditions the native AMF strains isolated from saline areas maintained a
higher symbiotic efficiency with maize plants than the collection strain.
Plant biomass production is an integrative measurement of plant performance
under many types of abiotic stress conditions and the symbiotic efficiency of AMF has
been measured in terms of plant growth improvement (see reviews by Evelin et al.,
2009; Ruiz-Lozano et al., 2012). In our experiment, maize plants inoculated with Ri
CdG and Ce CdG had the highest shoot dry biomass at all salinity levels, demonstrating
the higher symbiotic efficiency of these native AMF (Oliveira et al., 2005; Querejeta et
al., 2006). The growth of maize inoculated with Gi collect was similar to the non-
mycorrhizal plants, except at 100 mM NaCl, where it was lower. The latter can be
explained due to the high percentage of root colonization by this fungal strain that could
demand excessive carbohydrates from the plant. In fact plant growth responses to AMF
inoculation can range from parasitic to mutualistic (Klironomos, 2003). Sc CdG had a

139
Chapter 4

similar tendency; previous studies have reported that G. constrictum (= Septoglomus


constrictum) increased plant dry weight less than other AMF tested (Blaszkowski,
1993; Yu et al., 2010), suggesting a different symbiotic strategy to cope with abiotic
stresses rather than a parasitic behaviour. In any case, the positive effect of AM fungal
mycorrhization on growth was lower in root tissues than in shoot tissues, which is in
agreement with Hajiboland et al. (2010).
It has been proposed that mycorrhizal colonization enhances plant salt tolerance
by improving photosynthetic ability, water and nutrient uptake, ion balance and
osmolite concentration among others (Garg and Manchanda, 2009; Estrada et al., 2012;
Ruiz-Lozano et al., 2012). Salt injury can be avoided by maintaining proper osmotic
adjustment and ionic homeostasis. Salinity stress induces physiological drought in
plants, thus maintaining the water homeostasis is essential to alleviate the impact of
salinity on plant growth and crop yield (Dodd and Pérez-Alfocea, 2012). Indeed the
extensive hyphal network contributes to water and nutrient uptake because the AM
fungus can explore a larger soil volume (Evelin et al., 2012). Another method to
maintain a favourable gradient for water flow from soil into the roots is to decrease the
plant osmotic potential by active accumulation of organic ions or solutes (Ruiz-Lozano
et al., 2012). Proline is a major osmoprotectant osmolyte and in plants colonized by
AMF, it has been found to increase more than in non-AM plants at different salinity
levels (Sharifi et al., 2007; Talaat and Shawky, 2011). However, reports on the effect of
AM symbiosis on proline accumulation are somewhat contradictory and some authors
reported that non-MA plants accumulated more proline than AM plants (Rabie and
Almadini, 2005; Jahromi et al., 2008; Sheng et al., 2011). Our results did not show
significant differences in shoot proline concentration among fungal treatments. In the
root, proline content was several times higher than in the shoot and significantly
increased with the salinity levels in all treatments, except in Sc CdG. Higher levels of
proline in roots could be beneficial as these are the primary sites for water absorption
and must maintain osmotic balance between water absorbing root cells and external
media (Sharifi et al., 2007). In all, our results suggest that the enhanced salt tolerance in
AM maize plants was not due to differences in proline accumulation.
Under salt stress, plants not only accumulate some organic solutes like proline,
but also inorganic ions such as potassium to maintain osmotic adjustment (Yang et al.,
2009). In salinity conditions, plants increasingly accumulate Na+ ions which compete
with cellular K+ (Ruiz-Lozano et al., 2012). K+ functions cannot be replaced by Na+
ions, thus it is very important to maintain a proper ion homeostasis in terms of K+/Na+
ratio (Giri et al., 2007). Our results show a significant increase of K+ in the leaves of
maize plants inoculated with the three native AMF as compared to non-mycorrhizal
plants or plants inoculated with the collection fungus. Although in all the treatments Na+
accumulation increased with salinity, a higher K+/Na+ ratio was observed in the plants
inoculated with the three native AMF. Several authors have reported a decrease in Na+
and an increase in K+ concentrations in AM-inoculated plants (Garg and Manchanda,

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2009; Talaat and Shawky, 2011; Evelin et al., 2012). Results are also consistent with
Giri et al. (2007), who showed higher accumulation of K+ by mycorrhizal plants in
saline soils, thus maintaining a high K+/Na+ ratio which influences the ionic balance of
the cytoplasm or Na+ efflux from plants. Recently Hammer et al, (2011) demonstrated
that Rh. intraradices can selectively take up elements such as K+, Mg2+ and Ca2+ while
avoiding Na+ uptake. The latter indicates that the AM fungal mycelium might pre-select
nutrients for the plants. Moreover, as a significant proportion of elemental nutrient
uptake in plants occurs via mycorrhizal fungi, they help to alleviate the effects of the
excess of salts in the soil. Our results confirm that Se. constrictum also induced a higher
K+/Na+ ratio compared to non-mycorrhizal plants. In the roots, levels of Na+ were
always higher than in the leaves. It has been proposed that in AM-inoculated pants, Na+
might be kept inside root cell vacuoles and intraradical fungal hyphae to prevent the
allocation of Na+ to the shoots (Cantrell and Linderman, 2001). Plants inoculated with
Ce CdG had the lowest Na+ concentration at 66 and 100 mM of NaCl, being the most
efficient fungus in terms of avoiding Na+ uptake. Hammer et al. (2011) found different
concentrations and distributions of Na+ and Cl- within the fungal tissue and they
hypothesized that AMF exclude Na+ but include Cl-. Our results showed that all
treatments enhanced Cl- concentration as salinity in the growing medium increased.
Mardukhi et al. (2011) proposed that mycorrhizal plants had no control on plant Cl-
uptake. Thus the alleviating effect of AMF on plant growth under salinity stress is more
related to Na+ than Cl- uptake. Moreover, for maize, Na+ causes higher ion toxicity than
Cl- (Fortmeier and Schubert, 1995).
The previous results prompted us to hypothesize that AMF may have regulated
the expression of plant genes encoding for ion transporters. It is well documented that
overexpression of Na+/H+ and K+/H+ antiporters improve salt tolerance in plants (Zhang
et al., 2001; Rodriguez-Rosales et al., 2008). However, scarce information is available
on the possible regulation by the AM symbiosis of plant genes involved in ion
homeostasis. Until now, only Ouziad et al. (2006) have studied the effect of AM
symbiosis on the expression of two Na+/H+ antiporters in tomato under salt stress
conditions, showing no regulation of these genes by the AM symbiosis. Nevertheless,
we studied the expression of three genes involved in Na+ and K+ transport in order to
get some clues on molecular mechanisms involved in the enhanced tolerance of
mycorrhizal plants to salinity stress.
The SOS signalling pathway has been pointed out to have a major role
maintaining ion homeostasis by regulating Na+ and K+ transport at both the plasma
membrane and tonoplast (Zhu, 2002, 2003). AKT family contributes to a major
potassium acquisition by plants and SKOR transports K+ to the shoots (Munns, 2005).
Based on that, we analyzed the expression of some of these maize genes in the roots of
the different treatments. The most important differences among treatments were
observed at 100 mM NaCl for the three genes, where plants inoculated with Se CdG or
with Ce CdG exhibited enhanced relative expression. In contrast, under non saline

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conditions, these plants always showed reduced expression as compared to non-


mycorrhizal plants. These results correlate with the higher K+ and lower Na+
concentrations found in shoot tissues of maize plants. Thus we suggest that AMF can
affect plant ion transport via modification of gene expression. Moreover, different
species of AMF differ in the gene transport efficiency and native-AMF have better
regulation of these genes, thus enhancing plant salt tolerance.
Summarizing, the tolerance of maize to salt stress was enhanced by the three
native AMF more than by the collection one. Based on our results and on existing
literature, a major point for salt tolerance in mycorrhizal maize plants is the
improvement of plant nutrition and maintenance of ionic homeostasis. We showed
selective regulation by AMF of plant ion uptake and accumulation with subsequent
effects on the K+/Na+ ratio, correlating with plant transporters gene expression. The
more effective AMF were Cl. etunicatum CdG and Se. constrictum CdG. However, also
the native Rh. intraradices had a better ability to alleviate the inhibitory effect of salt
stress than the collection Rh. intraradices strain. The characterization of ion transporters
of these salt tolerant fungi should be the next step in understanding the molecular
mechanisms of salt tolerance acquired by AM symbiosis. The results obtained open
important possibilities for sustainable agricultural practices in salinized soils in order to
increase crop performance and yield production worldwide.

Acknowledgements

This work was financed by two research projects supported by Junta de Andalucía
(Spain). Projects P06-CVI-01876 and P11-CVI-7107. We thank Sonia Molina for
technical assistance and Domingo Álvarez (curator of the EEZ germplasm collection),
for taking care of the native AMF inocula. We also thank Dr. Jean Charles Isner for
essential support in primer design.

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References

Al-Karaki GN, Hammad R. 2001. Mycorrhizal influence on fruit yield and mineral
content of tomato grown under salt stress. Journal of Plant Nutrition 24, 1311-1323.
Aliasgharzadeh N, Rastin NS, Towfighi H, Alizadeh A. 2001. Occurrence of
arbuscular mycorrhizal fungi in saline soils of the Tabriz Plain of Iran in relation to
some physical and chemical properties of soil. Mycorrhiza 11, 119-122.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment
search tool. Journal of Molecular Biology 215, 403-410.
Aroca R, Porcel R, Ruíz-Lozano JM. 2007. How does arbuscular mycorrhizal
symbiosis regulate root hydraulic properties and plasma membrane aquaporins in
Phaseolus vulgaris under drought, cold or salinity stresses? New Phytologist 173, 808-
816.
Barea JM, Pozo MJ, López-Ráez JM, Aroca R, Ruíz-Lozano JM, Ferrol N, Azcón
R, Azcón-Aguilar C. 2012. Arbuscular Mycorrhizas and their significance in
promoting soil-plant systems sustainability against environmental stresses In: Rodelas
B, Gonzalez-Lopez J, eds. Beneficial Plant-Microbial Interactions: Ecology and
Applications USA: Science Publishers, (In press).
Bates LS, Waldren RP, Teare ID. 1973. Rapid determination of free proline for water-
stress studies. Plant and Soil 39, 205-207.
Becker WN, Gerdemann JW. 1977. Glomus etunicatus sp. nov. Mycotaxon 6, 29-32.
Blaszkowski J. 1993. Effects of five Glomus spp. (Zygomycetes) on growth and
mineral nutrition of Triticum aestivum L. Acta Mycologica 28, 201-210.
Bligh EG, Dyer WJ. 1959. A rapid method of total lipid extraction and purification.
Canadian Journal of Biochemistry and Physiology 37, 911-917.
Brundrett M, Melville L, Peterson L. 1994. Practical methods in mycorrhizal
research. University of Guelph, Ontario, Canada: Mycologue Publications.
Cantrell IC, Linderman RG. 2001. Preinoculation of lettuce and onion with VA
mycorrhizal fungi reduces deleterious effects of soil salinity. Plant and Soil 233, 269-
281.
Carvalho LM, Correia PM, Martins-Louçao MA. 2004. Arbuscular mycorrhizal
fungal propagules in a salt marsh. Mycorrhiza 14, 165-170.
Cataldo DA, Haroon M, Schrader LE, Young VL. 1975. Rapid colorimetric
determination of nitrate in plant tissue by nitration of salicylic acid. Communications in
Soil Science and Plant Analysis 6, 71-80.
Chen Z, Zhou MX, Newman IA, Mendham NJ, Zhang GP, Shabala S. 2007.
Potassium and sodium relations in salinised barley as a basis of differential salt
tolerante. Functional Plant Biology 34, 150-162.
Demidchik V, Maathuis FJM. 2007. Physiological roles of nonselective cation
channels in plants: from salt stress to signalling and development. New Phytologist 175,
387-404.

143
Chapter 4

Diatloff E, Rengel Z. 2001. Compilation of simple spectrophotometric techniques for


the determination of elements in nutrient solutions. Journal of Plant Nutrition 24, 75-
86.
Dodd IC, Pérez-Alfocea F. 2012. Microbial amelioration of crop salinity stress.
Journal of Experimental Botany 63, 3415-3428.
Estrada B, Barea JM, Aroca R, Ruiz-Lozano JM. 2012. A native Glomus
intraradices strain from a Mediterranean saline area exhibits salt tolerance and
enhanced symbiotic efficiency with maize plants under salt stress conditions. Plant and
Soil (In press) doi: 10.1007/s11104-012-1409-y
Evelin H, Giri B, Kapoor R. 2012. Contribution of Glomus intraradices inoculation to
nutrient acquisition and mitigation of ionic imbalance in NaCl-stressed Trigonella
foenum-graecum. Mycorrhiza 22, 203-217.
Evelin H, Kapoor R, Giri B. 2009. Arbuscular mycorrhizal fungi in alleviation of salt
stress: a review. Annals of Botany 104, 1263-1280.
FAO. 2005. Global Network on Integrated Soil Management for Sustainable Use of
Salt-affected Soils: Rome, Italy: FAO Land and Plant Nutrition Management Service.
http://www.fao.org/ag/agl/agll/spush.
Feng G, Zhang FS, Li XL, Tian CY, Tang C, Rengel Z. 2002. Improved tolerance of
maize plants to salt stress by arbuscular mycorrhiza is related to higher accumulation of
soluble sugars in roots. Mycorrhiza 12, 185-190.
Ferrol N, Calvente R, Cano C, Barea JM, Azcón-Aguilar C. 2004. Analysing
arbuscular mycorrhizal fungal diversity in shrub-associated resource islands from a
desertification-threatened semiarid Mediterranean ecosystem. Applied Soil Ecology 25,
123-133.
Fortmeier R, Schubert S. 1995. Salt tolerance of maize (Zea mays L.): the role of
sodium exclusion. Plant, Cell & Environment 18, 1041-1047.
Garg N, Manchanda G. 2009. Role of arbuscular mycorrhizae in the alleviation of
ionic, osmotic and oxidative stresses induced by salinity in Cajanus cajan (L.) Millsp
(pigeonpea). Journal of Agronomy and Crop Science 195, 110-123.
Giovannetti M, Mosse B. 1980. Evaluation of techniques for measuring vesicular
arbuscular mycorrhizal infection in roots. New Phytologist 84, 489-500.
Giri B, Kapoor R, Mukerji KG. 2007. Improved tolerance of Acacia nilotica to salt
stress by arbuscular mycorrhiza, Glomus fasciculatum may be partly related to elevated
K/Na ratios in root and shoot tissues. Microbial Ecology 54, 753-760.
Graham JH, Drouillard DL, Hodge NC. 1996. Carbon economy of sour orange in
response to different Glomus spp. Tree Physiology 16, 1023-1029.
Greenway H, Munns R. 1980. Mechanisms of salt tolerance in nonhalophytes Annual
Review of Plant Physiology 31, 149-190.
Hajiboland R, Aliasgharzadeh N, Laiegh SF, Poschenrieder C. 2010. Colonization
with arbuscular mycorrhizal fungi improves salinity tolerance of tomato (Solanum
lycopersicum L.) plants. Plant and Soil 331, 313-327.

144
Chapter 4

Hajlaoui H, Ayeb NE, Garrec JP, Denden M. 2010. Differential effects of salt stress
on osmotic adjustment and solutes allocation on the basis of root and leaf tissue
senescence of two silage maize (Zea mays L.) varieties. Industrial Crops and Products
31, 122-130.
Hammer E, Nasr H, Pallon J, Olsson P, Wallander H. 2011. Elemental composition
of arbuscular mycorrhizal fungi at high salinity. Mycorrhiza 21, 117-129.
Jahromi F, Aroca R, Porcel R, Ruiz-Lozano JM. 2008. Influence of salinity on the In
vitro development of Glomus intraradices and on the In vivo physiological and
molecular responses of mycorrhizal lettuce plants. Microbial Ecology 55, 45-53.
Khan MA, Shirazi MU, Ali M, Mumtaz S, Sherin A, Ashraf MY. 2006.
Comparative performance of some wheat genotypes growing under saline water.
Pakistan Journal of Botany 38, 1633-1639.
Klironomos JN. 2003. Variation in plant response to native and exotic arbuscular
mycorrhizal fungi. Ecology 84, 2292-2301.
Koske RE, Tessier B. 1983. A convenient, permanent slide mounting medium.
Mycological Society of America Newsletter 34, 59.
Krüger M, Krüger C, Walker C, Stockinger H, Schussler A. 2012. Phylogenetic
reference data for systematics and phylotaxonomy of arbuscular mycorrhizal fungi from
phylum to species level. New Phytologist 193, 970-984.
Lambers H. 2003. Introduction, dry land salinity: a key environmental issue in
Southern Australia. Plant and Soil 257, 5-7.
Lee J, Lee S, Young JPW. 2008. Improved PCR primers for the detection and
identification of arbuscular mycorrhizal fungi. FEMS Microbiology Ecology 65, 339-
349.
Lutts S, Kinet JM, Bouharmont J. 1996. Effects of salt stress on growth, mineral
nutrition and proline accumulation in relation to osmotic adjustment in rice (Oryza
sativa L.) cultivars differing in salinity resistance. Plant Growth Regulation 19, 207-
218.
Maas EV, Hoffman GJ. 1977. Crop salt tolerance current assessment. Journal of
Irrigation and Drainage Division, American Society of Civil Engineers 103, 115-134.
Maathuis FJM, Amtmann A. 1999. K+ nutrition and Na+ toxicity: the basis of cellular
K+/Na+ ratios. Annals of Botany 84, 123-133.
Mardukhi B, Rejali F, Daei G, Ardakani MR, Malakouti MJ, Miransari M. 2011.
Arbuscular mycorrhizas enhance nutrient uptake in different wheat genotypes at high
salinity levels under field and greenhouse conditions. Comptes Rendus Biologies 334,
564-571.
Mejía D. 2003. Maize: Post-Harvest Operations. Rome: AGST-FAO.
Munns R. 2005. Genes and salt tolerance: bringing them together. New Phytologist
167, 645-663.
Oehl F, Sieverding E, Palenzuela J, Ineichen K, Silva GA. 2011. Advances in
Glomeromycota taxonomy and classification. IMA Fungus 2, 191-199.

145
Chapter 4

Oliveira RS, Vosátka M, Dodd JC, Castro PML. 2005. Studies on the diversity of
arbuscular mycorrhizal fungi and the efficacy of two native isolates in a highly alkaline
anthropogenic sediment. Mycorrhiza 16, 23-31.
Ouziad F, Wilde P, Schmelzer E, Hildebrandt U, Bothe H. 2006. Analysis of
expression of aquaporins and Na+/H+ transporters in tomato colonized by arbuscular
mycorrhizal fungi and affected by salt stress. Environmental and Experimental Botany
57, 177-186.
Phillips JM, Hayman DS. 1970. Improved procedure of clearing roots and staining
parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection.
Transactions of the British Mycological Society 55, 159-161.
Pitman M, Läuchli A. 2002. Global impact of salinity and agricultural ecosystems. In:
Läuchli A, Lüttge U, eds. Salinity: environment-plants-molecules. Netherlands: Kluwer
Academic Publishers, 3-20.
Querejeta JI, Allen MF, Caravaca F, Roldán A. 2006. Differential modulation of
host plant δ13C and δ18O by native and nonnative arbuscular mycorrhizal fungi in a
semiarid environment. New Phytologist 169, 379-387.
Rabie GH, Almadini AM. 2005. Role of bioinoculants in development of salt-
tolerance of Vicia faba plants under salinity stress. African Journal of Biotechnology 4,
210-222.
Ramakers C, Ruijter JM, Lekane Deprez RH, Moorman AFM. 2003. Assumption-
free analysis of quantitative real-time polymerase chain reaction (PCR) data.
Neuroscience Letters 339, 62-66.
Rodriguez-Rosales MP, Jiang X, Gálvez FJ, Aranda MN, Cubero B, Venema K.
2008. Overexpression of the tomato K+/H+ antiporter LeNHX2 confers salt tolerance by
improving potassium compartmentalization. New Phytologist 179, 366-377.
Rodriguez HG, Roberts JKM, Jordan WR, Drew MC. 1997. Growth, water
relations, and accumulation of organic and inorganic solutes in roots of maize seedlings
during salt stress. Plant Physiology 113, 881-893.
Ruiz-Lozano JM, Collados C, Barea JM, Azcón R. 2001. Arbuscular mycorrhizal
symbiosis can alleviate drought-induced nodule senescence in soybean plants. New
Phytologist 151, 493-502.
Ruiz-Lozano JM, Porcel R, Azcón R, Aroca R. 2012. Regulation by arbuscular
mycorrhizae of the integrated physiological response to salinity in plants: new
challenges in physiological and molecular studies. Journal of Experimental Botany 63,
4033-4044.
Sannazzaro AI, Ruiz OA, Alberto EO, Menendez AB. 2006. Alleviation of salt stress
in Lotus glaber by Glomus intraradices. Plant and Soil 285, 279-287.
Schenk NC, Smith GS. 1982. Additional new and unreported species of mycorrhizal
fungi (Endogonaceae) from Florida. Mycologia 74, 77-92.

146
Chapter 4

Sharifi M, Ghorbanli M, Ebrahimzadeh H. 2007. Improved growth of salinity-


stressed soybean after inoculation with salt pre-treated mycorrhizal fungi. Journal of
Plant Physiology 164, 1144-1151.
Sheng M, Tang M, Chen H, Yang B, Zhang F, Huang Y. 2008. Influence of
arbuscular mycorrhizae on photosynthesis and water status of maize plants under salt
stress. Mycorrhiza 18, 287-296.
Sheng M, Tang M, Zhang FF, Huang YH. 2011. Influence of arbuscular mycorrhiza
on organic solutes in maize leaves under salt stress. Mycorrhiza 21, 423-430.
Sieverding E. 1991. Vesicular-arbuscular mycorrhiza management in tropical
agrosystems. Eschborn, Friedland, Bremer, Rossdorf, Germany: TZ-
Verlagsgesellschaft, Technical Cooperation (GTZ).
Slabu C, Zörb C, Steffens D, Schubert S. 2009. Is salt stress of faba bean (Vicia faba)
caused by Na+ or Cl- toxicity? Journal of Plant Nutrition and Soil Science 172, 644-651.
Smith SE, Read DJ. 2008. Mycorrhizal Symbiosis, 3rd Ed. New York: Elsevier,
Academic Press.
Spain JL. 1990. Arguments for diagnoses based on unaltered wall structures.
Mycotaxon 38, 71-76.
Talaat NB, Shawky BT. 2011. Influence of arbuscular mycorrhizae on yield, nutrients,
organic solutes, and antioxidant enzymes of two wheat cultivars under salt stress.
Journal of Plant Nutrition and Soil Science 174, 283-291.
Teakle NL, Tyerman SD. 2010. Mechanisms of Cl- transport contributing to salt
tolerance. Plant, Cell & Environment 33, 566-589.
Trappe JM. 1977. Three new Endogonaceae: Glomus constrictus, Sclerocystis
clavispora and Acaulospora scrobiculata. Mycotaxon 6, 359-366.
Vandesompele J, de Preter K, Pattyn F, Poppe B, van Roy N, de Paepe A,
Speleman F. 2002. Accurate normalization of real-time quantitative RT-PCR data by
geometric averaging of multiple internal control genes. Genome Biology 3, 1-11.
White PJ, Broadley MR. 2001. Chloride in soils and its uptake and movement within
the plant: a review. Annals of Botany 88, 967-988.
Wilde P, Manal A, Stodden M, Sieverding E, Hildebrandt U, Bothe H. 2009.
Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt marshes.
Environmental Microbiology 11, 1548-1561.
Wu QS, Zou YN, He XH. 2010. Contributions of arbuscular mycorrhizal fungi to
growth, photosynthesis, root morphology and ionic balance of citrus seedlings under
salt stress. Acta Physiologiae Plantarum 32, 297-304.
Xu G, Magen H, Tarchitzky J, Kafkafi U. 2000. Advances in chloride nutrition.
Advances in Agronomy 68, 96-150.
Yamato M, Ikeda S, Iwase K. 2008. Community of arbuscular mycorrhizal fungi in a
coastal vegetation on Okinawa island and effect of the isolated fungi on growth of
sorghum under salt-treated conditions. Mycorrhiza 18, 241-249.

147
Chapter 4

Yang CW, Xu HH, Wang LL, Liu J, Shi DC, Wang GD. 2009. Comparative effects
of salt-stress and alkali-stress on the growth, photosynthesis, solute accumulation, and
ion balance of barley plants. Photosynthetica 47, 79-86.
Yu Y, Zhang S, Huang H, Wu N. 2010. Uptake of arsenic by maize inoculated with
three different arbuscular mycorrhizal fungi. Communication in Soil Science and Plant
Analysis 41, 735-743.
Zhang XH, Hodson I, Williams JP, Blumwald E. 2001. Engineering salt-tolerant
Brassica plants: characterization of yield and seed oil quality in transgenic plants with
increased vacuolar sodium accumulation. Proceedings of the National Academy of
Sciences, USA 98, 12832-12836.
Zhu JK. 2002. Salt and drought stress signal transduction in plants. Annual Review of
Plant Biology 53, 247-273.
Zhu JK. 2003. Regulation of ion homeostasis under salt stress. Current Opinion in
Plant Biology 6, 441-445.

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CHAPTER 5

Native arbuscular mycorrhizal fungi isolated from a saline habitat


improved maize antioxidant systems and plant tolerance to salinity

Submitted to Plant Science by Estrada B; Aroca R; Barea J.M and


Ruiz-Lozano J.M.
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Capítulo 5

Hongos formadores de micorrizas arbusculares aislados de hábitat


salinos mejoran el sistema antioxidante del maíz y su tolerancia a la
salinidad

Resumen

La alta salinidad del suelo es un serio problema para la producción agrícola porque la
mayoría de las plantas cultivadas son sensibles sal. Este es el caso de un cultivo tan
importante como el maíz. El estrés salino conduce a un estrés oxidativo secundario en
plantas y se ha demostrado en varias especies de plantas una correlación entre la
capacidad antioxidante y tolerancia a la salinidad. La capacidad antioxidante de la
planta puede ser potenciada por los hongos micorrícicos arbusculares (MA) y se ha
propuesto que la simbiosis MA es más eficaz con especies nativas que con exóticas. Por
lo tanto, se investigó si hongos nativos MA aislados de un ambiente salino pueden
ayudar a las plantas de maíz a superar el estrés salino mejor que hongos MA de
colección y si la protección frente al estrés oxidativo está implicada en este efecto. Las
plantas de maíz inoculadas con los tres hongos nativos MA mostraron una mayor
eficiencia del fotosistema II y la conductancia estomática, lo que sin duda contribuyó a
reducir la fotorrespiración y la producción de ROS. De hecho, la acumulación de
peróxido de hidrógeno, el daño oxidativo a lípidos y la pérdida de electrolitos en la
membrana de estas plantas MA fueron significativamente más bajos que en las no
micorrizadas o en plantas inoculadas con el hongo MA de colección. La activación de
enzimas antioxidantes como la superóxido dismutasa o catalasa también contribuyeron
a estos efectos.

Palabras clave: sistema antioxidante, micorriza arbuscular, tolerancia de plantas,


salinidad, adaptado a la salinidad

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Native arbuscular mycorrhizal fungi isolated from a saline habitat


improved maize antioxidant systems and plant tolerance to salinity

Beatriz Estrada, Ricardo Aroca, José Miguel Barea and Juan Manuel Ruiz-
Lozano*

Departamento de Microbiología del Suelo y Sistemas Simbióticos. Estación


Experimental del Zaidín (CSIC). Profesor Albareda nº 1, 18008 Granada, Spain.

* Corresponding author: Dr. Juan Manuel Ruiz-Lozano.


e-mail: juanmanuel.ruiz@eez.csic.es
Telph. + 34 958 181600
Fax: + 34 958 129600

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Abstract

High soil salinity is a serious problem for crop production because most of the
cultivated plants are salt sensitive. This is the case of a so important crop such as maize.
Salinity stress leads to secondary oxidative stress in plants and a correlation between
antioxidant capacity and salt tolerance has been demonstrated in several plant species.
The plant antioxidant capacity may be enhanced by arbuscular mycorrhizal fungi
(AMF) and it has been proposed that AM symbiosis is more effective with native than
with exotic AMF species. Thus, we investigated whether native AMF isolated from a
saline environment can help maize plants to overcome salt stress better than AMF from
collection and whether protection against oxidative stress is involved in such an effect.
Maize plants inoculated with three native AMF showed higher efficiency of
photosystem II and stomatal conductance, which surely contributed to decrease
photorespiration and ROS production. Indeed, the accumulation of hydrogen peroxide,
the oxidative damage to lipids and the membrane electrolyte leakage in these AM plants
were significantly lower than in non-mycorrhizal plants or in plants inoculated with the
collection AMF. The activation of antioxidant enzymes such as superoxide dismutase or
catalase also accounted for these effects.

Key words: antioxidant system, arbuscular mycorrhiza, plant tolerance, salinity, salt-
adapted

1. Introduction

Maize (Zea mays L.) is one of the most important crops both for human and
animal consumption. This crop is cultivated on more than 142 million ha of land
worldwide and it is estimated to produce around 913 million tonnes of grain in the
period 2012/2013 [1], accounting for one third of the total global grain production [2].
In a climate change scenario, one expected threat is the increase in land salinization.
Over 6% of the world’s land is affected by salinity and its extent is increasing regularly
throughout the world [3], causing global agricultural losses equivalent to an estimated
US 12 billion a year [4]. Although salinity in soils may occur naturally, inappropriate
cultivation practices are also contributing to the salinization of the rhizosphere [5].
Nowadays, high salts in the soils are a very serious problem for crop production because
most of the cultivated plants are sensitive to salt stress (glycophytes) [6]. This is the
case of maize, which is particularly vulnerable to salinity [7]. Thus land salinization is a
major global issue because of its adverse impact on agricultural productivity,
sustainability and as a threat for food supply [8]. In arid and semi-arid regions, the issue

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is aggravated due to poor soil management practices together with limited rainfall, high
evapotranspiration, and high temperature rates [9].
Salinity stress leads to secondary oxidative stress in plants [10]. The latter is
produced when pathways are uncoupled in the metabolism of plants and electrons, with
high-energy state, are transferred to molecular oxygen to form reactive oxygen species
(ROS) [10]. ROS are normally produced at a low level in organelles such as
chloroplasts, mitochondria and peroxisomes. However, under salt-stress conditions,
their production dramatically increases due to the accumulation of NADPH and ATP
that cannot be consumed [11]. This modifies the balance between the formation and
removal of ROS species [12]. Singlet oxygen (1O2), superoxide radical (O2•−), hydroxyl
radical (OH•) and hydrogen peroxide (H2O2) can seriously disrupt normal metabolism
through denaturalization of proteins, mutation of DNA and lipid peroxidation [13].
Thus, plants need to have appropriate detoxification systems to allow rapid removal of
these compounds. They constantly sense the level of ROS and reprogramme their gene
expression to respond to changes in their environment [13]. The most common
mechanism to detoxify ROS produced during salt-stress response is the induction of
ROS-scavenging enzymes, such as superoxide dismutase (SOD) and catalase (CAT)
[14]. SOD converts O2•− to H2O2 and then CAT converts H2O2 to water and molecular
oxygen in peroxisomes. A correlation between antioxidant capacity and NaCl tolerance
has been demonstrated in several plant species [15]. Despite the several mechanisms
developed by plants to detoxify the excess of ROS production, most of the cultivated
plants are glycophytes and under high salts in the soil they cannot cope with the extra
production of ROS [6].
Fortunately, a number of beneficial soil microorganisms, particularly arbuscular
mycorrhiza fungi (AMF), help plants to cope with abiotic stress conditions [16]. AMF,
belonging to the phylum Glomeromycota, are considered the oldest group of organisms
living in symbiosis with terrestrial plants [17]. They are fundamental for soil fertility,
both in natural and agricultural ecosystems and most land plants are colonized by these
fungi [18]. AMF are widely found in saline soils [19-20]. In fact, several authors have
demonstrated the beneficial effect of AMF in growth and productivity under salt stress
conditions on glycophytes and halophytes, decreasing plant yield loses (reviewed by
[11, 21]. Among other mechanisms, some studies have demonstrated that AM
symbiosis enhances the activities of antioxidant enzymes, helping plants to alleviate salt
stress [22-23]. Most studies have focused in the use of collection species as AMF
inoculum [21]. However several studies showed that native AMF can perform better in
soils from which they are isolated: agricultural systems [24], polluted soils [25] and
semiarid degraded soils [26] among others. According to this, some authors reported
that AM symbiosis, is more effective with native than with exotic AMF species [27-29].
However, the mechanisms that allow AM plants to have higher salinity tolerance are far
from being understood [11]. The isolation and study of AMF isolated from a saline area

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will contribute to elucidate the ecophysiology of AMF under such stress conditions
[25].
The aim of the present study was to investigate whether native AMF isolated
from a saline environment can help maize plants to overcome the negative effects of salt
stress better than AMF from collection and whether protection against oxidative stress
is involved in such an effect. Plant physiological and biochemical parameters related to
the oxidative status were determined in non-AM maize plants and in maize plants
inoculated with different AMF isolates after a salt stress period.

2. Materials and methods

2.1. Identification of the mycorrhizal strains isolated from Cabo de Gata Natural Park

AM fungal spores were separated from the soil samples by a wet sieving process
[30]. The morphological spore characteristics and their subcellular structures were
described from a specimen mounted in: polyvinyl alcohol-lactic acid-glycerine (PVLG)
[31]; a mixture of PVLG and Melzer’s reagent [32]; a mixture of lactic acid to water at
1:1; Melzer’s reagent; and water [33]. For identification of the AMF species, spores
were then examined using a compound microscope at up to 400-fold magnification as
described for glomeromycotean classification by Oehl et al. [34]. The species were
identified based on its spore morphology as a Rhizophagus intraradices [35],
Claroideoglomus etunicatum [36] and Septoglomus constrictum [37].
In addition to the morphological identification, a molecular identification was
also carried out. For that, spores isolated from the bait cultures of each fungal strain
were surface-sterilized with chloramine T (2%) and streptomycin (0.02%) and crushed
with a sterile disposable micropestle in 40 μL milli-Q water [38]. A two-step PCR was
conducted to amplify the AM fungal DNA from the spores. The first PCR step was
performed with the universal eukaryote primers NS1 and NS4 region of the small
subunit ribosomal gene and the second with the specific AM fungal primers AML1 and
AML2 [39]. The amplified DNA was purified using the Ilustra™ GFX™ PCR DNA
and Gel Band Purification Kit (GE Helthcare, UK). DNA fragments were sequenced on
an automated DNA sequencer (Perkin-Elmer ABI Prism 373). Sequence data were
compared to gene libraries (EMBL and GenBank) using BLAST program [40].
The BLAST analysis unambiguously placed Rhizophagus intraradices as the
closest relative of our Rhizophagus intraradices CdG strain, with sequence accession
number FR750209 [41] having a 99% identity. Septoglomus constrictum was the closest
relative to our Se. constrictum CdG strain, with sequence accession number FR750212
[41] having a 99% identity. Finally, Claroideoglomus etunicatum was the closest
relative of our Cl. etunicatum CdG strain, with sequence accession number FR750216.1
[41] having also a 99% identity. The AM fungal strains have been incorporated to the

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collection of Zaidin Experimental Station, Granada, Spain, under accession numbers


EEZ 195, EEZ 196 and EEZ 163, respectively.

2.2. Experimental design

The experiment consisted of a randomized complete block design with five


inoculation treatments: (1) non-mycorrhizal control plants, (2) plants inoculated with
the model AM fungus Rh. intraradices (Ri collect), reproduced at the collection of the
Zaidin Experimental Station (isolate EEZ01), (3) plants inoculated with the AM fungal
strain Rh. intraradices isolated from Cabo de Gata Natural Park (Ri CdG), (4) plants
inoculated with the AM fungal strain Se. constrictum isolated from CdG (Sc CdG) and
(5) plants inoculated with the AM fungal strain Cl. etunicatum isolated from CdG (Ce
CdG). There were 30 replicates of each inoculation treatment, totalling 150 pots (one
plant per pot), so that ten of each microbial treatment were grown under non-saline
conditions throughout the entire experiment, while ten pots per treatment were
subjected to 66 mM of NaCl and the remaining ten pots per treatment were subjected to
100 mM of NaCl.

2.3. Soil and biological materials

Loamy soil was collected from Granada province (Spain, 36º59’34’’N;


3º34’47’’W), sieved (5 mm), diluted with quartz-sand (<2 mm) (1:1, soil:sand, v/v) and
sterilized by steaming (100ºC for 1 h on 3 consecutive days). The original soil had a pH
of 8.2 [measured in water 1:5 (w/v)]; 1.5 % organic matter, nutrient concentrations (g
kg-1): N, 1.9; P, 1 (NaHCO3-extractable P); K, 6.9. The electrical conductivity of the
original soil was 0.5 dS m-1.
Three seeds of maize (Zea mays. L) were sown in pots containing 900 g of the
same soil/sand mixture as described above and thinned to one seedling per pot after
emergence.

2.4. Inoculation treatments

Mycorrhizal inoculum was bulked in an open-pot culture of Zea mays L. and


consisted of soil, spores, mycelia and infected root fragments. The AM species used
were three strains isolated from Cabo de Gata Natural Park (Almería, Spain):
Rhizophagus intraradices (previously named Glomus intraradices), Septoglomus
constrictum and Claroideoglomus etunicatum. A Rhizophagus intraradices strain from
our culture collection was also used. Appropriate amounts of each inoculum containing
about 700 infective propagules (according to the most probable number test), were
added to the corresponding pots at sowing time just below maize seeds. Non-
mycorrhizal control plants received the same amount of autoclaved mycorrhizal inocula

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together with a 10 ml aliquot of a filtrate (< 20 μm) of the AM inocula in order to


provide a general microbial population free of AM propagules.

2.5. Growth conditions

The experiment was carried out under glasshouse conditions with temperatures
ranging from 19 to 25ºC, 16/8 light/dark period, and a relative humidity of 50-60%. A
photosynthetic photon flux density of 800 μE m-2 s-1 was measured with a light meter
(LICOR, Lincoln, NE, USA, model LI-188B). Water was supplied daily to the entire
period of plant growth to avoid any drought effect. Plants were established for 45 days
prior to salinization to allow adequate plant growth and symbiotic establishment. Three
concentrations (0, 66, and 100 mM NaCl) of saline solution were reached in the soil
substrate by adding appropriate dilutions of a stock 2 M saline solution. The
concentration of NaCl in the soil was increased gradually on alternative days to avoid an
osmotic shock. It took 8 days, to reach the desired 66 and 100 mM NaCl levels. Plants
were maintained under these conditions for additional 30 days.

2.6. Symbiotic development

The percentage of mycorrhizal root infection in maize plants was estimated by


visual observation of fungal colonization after clearing washed roots in 10% KOH and
staining with 0.05% trypan blue in lactic acid (v/v), as described by Phillips and
Hayman [42]. The extent of mycorrhizal colonization was calculated according to the
gridline intersect method [43].

2.7. Shoot biomass production

At harvest (75 days after planting), the shoot and root system were separated and the
shoot dry weight was measured after drying in a forced hot-air oven at 70ºC for two
days.

2.8. Photosynthetic efficiency

The efficiency of photosystem II was measured with FluorPen FP100 (Photon


Systems Instruments, Brno, Czech Republic), which allows a non-invasive assessment
of plant photosynthetic performance by measuring chlorophyll a fluorescence. FluorPen
quantifies the quantum yield of photosystem II as the ratio between the actual
fluorescence yield in the light-adapted state (FV‘) and the maximum fluorescence yield
in the light-adapted state (FM‘), according to [44]. Measurements were taken in the
second youngest leaf of ten different plants of each treatment.

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2.9. Stomatal conductance

Stomatal conductance was measured two hours after light turned on by using a
porometer system (Porometer AP4, Delta-T Devices Ltd, Cambridge, UK) following
the user manual instructions. Stomatal conductance measurements were taken in the
second youngest leaf from five different plants from each treatment.

2.10. Relative electrolyte leakage

The electrolyte leakage was calculated on the third leaf of each maize plant from
a leaf sample of 3 x 1.5 cm as described by Verslues et al. [45]. The initial conductivity
(C0) was measured with a conductivity metre COND 510 (XS Instruments; OptoLab,
Milan, Italy) after subjecting the samples to incubation at 25ºC in 10 ml de-ionized
water overnight with continuous shaking at 100 rpm. The samples were then autoclaved
at 121ºC for 20 min. Final conductivity (CF) was measured after the samples had cooled
down to room temperature. The conductivity of distilled water was also measured and
referred as Cw. The percentage of electrolyte leakage was calculated as follows:
[(C0 – Cw) / (CF – Cw)] x 100.

2.11. Oxidative damage to lipids

Lipid peroxides were extracted by grinding 500 mg of leaves and roots with an
ice-cold mortar and 6 ml of 100 mM potassium phosphate buffer (pH 7). Homogenates
were filtered through one Miracloth layer and centrifuged at 15,000 × g for 20 min. The
chromogen was formed by mixing 200 μl of supernatants with 1 ml of a reaction
mixture containing 15% (w/v), trichloroacetic acid (TCA), 0.375% (w/v) 2-
thiobarbituric acid (TBA), 0.1% (w/v) butyl hydroxytoluene, 0.25N HCl and then
incubating the mixture at 100ºC for 30 min [46]. After cooling at room temperature,
tubes were centrifuged at 800 × g for 5 min and the supernatant was used for
spectrophotometric reading at 532 nm. Lipid peroxidation was estimated as the content
of 2-thiobarbituric acid-reactive substances (TBARS) and expressed as equivalents of
malondialdehyde (MDA) according to Halliwell and Gutteridge [47]. The calibration
curve was made using MDA in the range of 0.1-10 nmol. A blank for all samples was
prepared by replacing the sample with extraction medium, and controls for each sample
were prepared by replacing TBA with 0.25N HCl. In all cases, 0.1% (w/v) butyl
hydroxytoluene was included in the reaction mixtures to prevent artifactual formation of
TBARS during the acid-heating step of the assay.

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2.12. Hydrogen peroxide content

Hydrogen peroxide content in leaves and roots were determined by Patterson et


al. [48] method, with slight modifications as described previously by Aroca et al. [49].
Two hundred and fifty milligrams of shoot fresh weight was homogenized in a cold
mortar with 5 ml 5% (w/v) TCA containing 0.1 g of activated charcoal and 1% (w/v)
PVPP. The homogenate was centrifuged at 18,000 x g for 10 min. The supernatant was
filtered through a Millipore filter (0.22 μm). A volume of 1.2 ml of 100 mM potassium
phosphate buffer (pH 8.4) and 0.6 ml of the colorimetric reagent were added to 130 μl
of the supernatant. The colorimetric reagent was freshly made by mixing 1:1 (v/v) 0.6
mM potassium titanium oxalate and 0.6 mM 4-2 (2-pyridylazo) resorcinol (disodium
salt). The samples were incubated at 45ºC for 1 h and the absorbance at 508 nm was
recorded. The blanks were made by replacing sample extract by 5% TCA.

2.13. Antioxidant enzymes activities

Enzyme extraction was done as described before by Aroca et al. [50] with slight
modifications. Five hundred mg of leaf and root fresh tissues were homogenized
separately in a cold mortar with 4 ml of 100 mM phosphate buffer (pH 7.0) containing
0.1 mM DTPA (diethylenetriamine pentaacetic acid; a metal chelating agent) and 40 mg
PVPP (polyvinylpolypyrrolidone), which removes phenolics and alkaloids from plant
extracts, avoiding interference with spectrophotometric measurements and enhancing
enzyme stability. The homogenate was centrifuged at 20,000 x g for 20 min at 4ºC. The
supernatant was separated and used to determine the activity of antioxidant enzymes.
All enzymatic activities were measured in a spectrophotometer InfiniteR 200 PRO series
(Tecan Trading AG, Switzerland) at 25 °C. Total protein was assayed by the method
described by Bradford [51].
Total superoxide dismutase (SOD) (EC 1.15.1.1) activity was measured
according to Becana et al. [52]. One unit of SOD activity (U) was defined as the amount
of enzyme which produced a 50% inhibition of nitroblue tetrazolium (NBT) reduction.
The reaction mixture (1,9 ml) contained 50 mM phosphate buffer (pH 7.8), 14,3 M
methionine, 82,5 μM NBT, 2,2 μM riboflavin and 100 μl enzyme extract. Riboflavin
was added last and tubes were shaken and illuminated with fluorescent light. The
reaction was allowed to proceed for 10 min until the colour of the blank shifted to dark
violet. Absorbance of the reaction mixture was read at 560 nm.
Catalase (CAT) (EC1.11.1.6) activity was assayed as described by [53].
Consumption of H2O2 (extinction coefficient of 39.6 mM-1cm-1) at 240 nm for 1 min
was monitored. The reaction mixture consisted of 50 mM phosphate buffer (pH 7.0)
containing 10 mM H2O2 and 5 μl of enzyme extract in a 205 μl volume.

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2.14. Statistical Analysis

Statistical analysis was performed using SPSS 19.0 statistical program (SPSS
Inc., Chicago, IL, USA) performing first a one-way ANOVA followed by the Tukey
test with P< 0.05 as the significance cut-off. Two independent statistical analyses were
carried out: the first to analyze data from the different AMF treatments within each
saline level and the second one to analyze data from each fungal treatment at increasing
salinity.

3. Results

3.1. Symbiotic development and shoot biomass production

Root AM colonization increased by salinity in plants colonized by all the fungus


except those colonized by Ri CdG, which showed the lowest values of root colonization
at all salinity levels (Table 1). Under both saline conditions (66 and 100 mM NaCl) Ri
collect showed the highest rate of root length colonization (Table 1). The other two
fungi (Sc CdG and Ce CdG) showed intermediate colonization capacity (Table 1).
The shoot biomass production in all treatments was negatively affected by the
increase of salt application. In the case of non-mycorrhizal plants the decrease was not
significant (Table 1). In any case, at each saline level, both Ri CdG and Ce CdG,
enhanced maize shoot biomass as compared to the non-mycorrhizal plants, while Ri
collect and Sc CdG did not (Table 1).

3.2. Stomatal conductance

Under non-saline conditions no differences in stomatal conductance were


observed among inoculation treatments (Fig. 1A). Salinity application decreased
stomatal conductance in all treatments. However, at both saline levels, plants inoculated
with the three native AMF from Cabo de Gata showed higher stomatal conductance
than non-mycorrhizal plants or plants colonized by Ri collect (Fig. 1A).

3.3. Photosystem II efficiency

The application of 100 mM NaCl, decreased the efficiency of photosystem II in


maize plants as compared to plants cultivated at 0 mM NaCl. Under non-saline
conditions, photosynthetic efficiency was increased by inoculation with all the native
AMF from Cabo de Gata (Fig. 1B). Growing under 66 mM NaCl did not cause any
significant descent in photosynthetic efficiency, except in plants inoculated with Ce

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CdG (Fig. 1B). When plants were subjected to 100 mM NaCl all the mycorrhizal plants
showed higher photosynthetic efficiency than non-mycorrhizal plants, mainly plants
inoculated with Ri CdG and Ce CdG (Fig. 1B).

AMF NaCl AM root Shoot Dry


treatment (mM) colonization (%) Weight

0 0 4.03 ± 0.14 Ab
NM 66 0 3.95 ± 0.12 Ab
100 0 3.69 ± 0.11 Ab

0 67 ± 1.9 Ea 3.87 ± 0.10 Db


Ri collect 66 88.7 ± 2.0 Da 3.51 ± 0.14 DEb
100 84.3 ± 1.1 Da 3.17 ± 0.16 Ec

0 21 ± 5.8 Gc 5.07 ± 0.22 Ga


Ri CdG 66 19.7 ± 1.5 Gc 4.62 ± 0.17 GHa
100 23 ± 0.7 Gd 4.19 ± 0.11 Ha

0 34.7 ± 1.5 Kb 3.78 ± 0.31 Jb


Sc CdG 66 62.7 ± 4.5 Jb 3.64 ± 0.20 Jb
100 58.7 ± 1.1 Jc 2.90 ± 0.08 Kc

0 62.3 ± 2.3 Xa 4.97 ± 0.16 Wa


Ce CdG 66 65.3 ± 1.1 Xb 5.17 ± 0.11 Wa
100 77.3 ± 1.8 Wb 4.62 ± 0.10 Xa

Table 1. Percentage of mycorrhizal root colonization and shoot dry weight (g plant-1) in maize plants.
NM represents non-mycorrhizal control plants; Ri collect, plants inoculated with the collection
Rhizophagus intraradices strain; Ri CdG, plants inoculated with the native Rh. intraradices CdG strain;
Sc CdG, plants inoculated with the native Septoglomus claroideum CdG strain and Ce CdG, plants
inoculated with the native Claroideoglomus etunicatum CdG strain. Plants were subjected to 0, 66 or 100
mM NaCl. Different letters indicate significant differences (p < 0.05) among fungal treatments at each
salt level (a, b, c, d) or among salt levels for each AMF treatment: NM plants (A, B, C), Ri collect (D, E,
F), Ri CdG (G, H, I), Sc CdG (J, K, L) or Ce CdG (W, X, Y).

3.4. Relative electrolyte leakage

The application of 66 or 100 mM NaCl did not significantly affect the electrolyte
leakage of maize plants as compared to plants growing in the absence of salinity (Fig.
1C). However, at each saline level non-mycorrhizal plants had always the highest values
of electrolyte leakage, followed by plants inoculated with Ri collect. In contrast, maize
plants inoculated with any of the three native AMF always showed significantly lower
electrolyte leakage than control non-mycorrhizal plants (Fig. 1C).

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35 J

Stomatal conductance
A G a W A

(mmolH2Om-2s-1)
30 a D a a
25 a X X
H K a
H
20 B E a ab K a
b b B a NM
a
15 b F Ri collect
Ri CdG
10 c Sc CdG
Ce CdG
5
0 mM 66 mM 100 mM

G W
0.7
Photosynthetic efficiency

J G B
a a X
A D a
0.6 A D a JK H X
b b b
b b b a K a
E ab
0.5
b
B
0.4 c
0.3

0.2
0 mM 66 mM 100 mM

25
C
(%)

A
leakage(%)

A A D a
20 D
ElectrolyteLeakage

a D G J W a ab J W W
a G bc c b G J bc
15 b b b c c
c
Electrolyte

10

0
0 mM 66 mM 100 mM

Fig. 1. Stomatal conductance (mmol H2O m-2s-1) (A), efficiency of photosystem II (B) and electrolyte
leakage (C) in maize plants. Black bars represent non-mycorrhizal control plants (NM); grey bars, plants
inoculated with the collection Rhizophagus intraradices strain (Ri collect); white bars, plants inoculated
with the native Rh. intraradices CdG strain (Ri CdG); lined bars, plants inoculated with the native
Septoglomus claroideum CdG strain (Sc CdG) and dotted bars, plants inoculated with the native
Claroideoglomus etunicatum CdG strain (Ce CdG). Plants were subjected to 0, 66 or 100 mM NaCl.
Different letters indicate significant differences (p < 0.05) among fungal treatments at each salt level (a, b,
c, d) or among salt levels for each AMF treatment: NM plants (A, B, C), Ri collect (D, E, F), Ri CdG (G,
H, I), Sc CdG (J, K, L) or Ce CdG (W, X, Y).

3.5. Oxidative damage to lipids

The application of 66 or 100 mM NaCl increased lipid peroxidation in leaves of


non-mycorrhizal plants and plants inoculated with Ri collect and Ce CdG. Plants
inoculated with Ri CdG only enhanced lipid peroxidation at 100 mM NaCl, while plants

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Chapter 5

inoculated with Sc CdG showed no significant changes in this parameter as a


consequence of increasing salinity. When plants were grown at 66 or 100 mM NaCl, the
plants inoculated with the three native AMF from Cabo de Gata showed the lowest
values of lipid peroxidation in their leaves (Fig. 2A). In roots, the lipid peroxidation also
increased with increasing salinity, especially in non-mycorrhizal plants and in plants
inoculated with Ri collect at 100 mM NaCl. In contrast, in plants inoculated with any of
the three native AMF from Cabo de Gata, the increase in lipid peroxidation due to
salinity was less evident. In addition, these plants always exhibited lower lipid
peroxidation in roots than non-mycorrhizal plants or those inoculated with Ri collect
(Fig. 2B).
D A D
A
25 a a a W
a A
GH J X G J b
20 E b b
J b b b
a H
15 a Y
B a ab
b NM
10 Ri collect
Oxidative damage to lipids

Ri CdG
5
(nmol MDA g-1 DW)

Sc CdG
Ce CdG
0
0 mM 66 mM 100 mM

25 F
D B
20 A a
E a
a J W
15 F
D B WX G J b
B
a G b G b b
10 a X b b
b
b K b
5 b

0
0 mM 66 mM 100 mM

Fig. 2. Shoot (A) and root (B) oxidative damage to lipids in maize plants. See legend for Fig. 1.

3.6. Hydrogen peroxide accumulation

In leaves, the accumulation of hydrogen peroxide due to the increasing salinity


only rose in non-mycorrhizal plants and in plants colonized by Ri collect when
subjected to 100 mM NaCl (Fig. 3A). At this NaCl concentration the lowest value of
hydrogen peroxide was observed in leaves of plants colonized by Ri CdG (Fig. 3A). In
roots, the accumulation of hydrogen peroxide increased considerably in non-
mycorrhizal plants after exposure to 66 or 100 mM NaCl. Plants inoculated with Ri
collect and Ri CdG plants also enhanced this parameter as a consequence of increasing
salinity (Fig. 3B). However, at 66 and 100 mM NaCl, the accumulation of hydrogen
peroxide was lower in roots of plants colonized by any of the three native AMF from

164
Chapter 5

Cabo de Gata than in the non-mycorrhizal plants or plants colonized by Ri collect (at
100 mM NaCl) (Fig. 3B).

0.8 A
A D
0.7
a a J
0.6
E G
J B E G J G ab W
0.5 W
a W a a a a b ab
Hydrogen peroxide (μmol g-1 DW)

0.4 B ab ab ab
a
b NM
0.3 Ri collect
0.2 Ri CdG
Sc CdG
0.1 Ce CdG
0
0 mM 66 mM 100 mM

A
0.9 a B
0.8 A
0.7 D
a
0.6 D a
0.5 W
J W
ab
G J b G J W
0.4 B E
ab a b b b b b
0.3 ab ab H
0.2 b
0.1
0.0
0
0 mM 66 mM 100 mM

Fig. 3. Shoot (A) and root (B) hydroxide peroxide content in maize plants. See legend for Fig. 1.

3.7. Antioxidant enzyme activity

In non-mycorrhizal plants, leaf SOD activity increased considerably by both salt


treatments and it increased only slightly in Ri CdG and Ce CdG plants after application
of 100 mM NaCl (Fig. 4A). Plants inoculated with Ri collect or Sc CdG did not alter
significantly their leaf SOD activity by any of the salt treatments (Fig. 4A). When data
were analyzed within each salt level, no significant differences in leaf SOD activity
among inoculation treatments were observed at 66 or 100 mM NaCl, while at 0 mM
NaCl, three out the four mycorrhizal treatments exhibited higher SOD activity in their
leaves than the non-mycorrhizal plants (Fig. 4A). Root SOD activity increased by salt
application only in plants colonized by Ce CdG (at both 66 and 100 mM NaCl) and in
plants colonized by Ri CdG (at 100 mM NaCl) (Fig. 4B). Under 100 mM NaCl
conditions, plants colonized by Ce CdG showed the highest root SOD activity, followed
by plants colonized by Ri CdG and Sc CdG. Non-mycorrhizal plants and those
colonized by Ri collect had the lowest values of root SOD activity under the highest salt
concentration treatment (Fig. 4B).

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Chapter 5

J W
12 A
a D a
A J A J
10 a
DE X a a WX a E G a
8 ab GH a a
H b a
a
6
SOD activity (U min-1mg-1Prot)

bc NM
4 B Ri collect
Ri CdG
2 c Sc CdG
Ce CdG
0
0 mM 66 mM 100 mM

W
25 a B
X G J
20 J
a b b
D A D a
15 H J Y
A a a a a ab ab A
H D
a c
10 b c

0
0 mM 66 mM 100 mM

Fig. 4. Shoot (A) and root (B) SOD activity in maize plants. See legend for Fig. 1.

The catalase activity in shoots resulted differently affected by salt application


depending on the microbial treatment. Thus, non-mycorrhizal plants and plants
inoculated with Ri collect enhanced transiently CAT activity after application of 66 mM
NaCl, but they almost recovered this activity to initial values after application of 100
mM NaCl. (Fig. 5A). Plants inoculated with Ri CdG only increased CAT activity when
the salt applied reached 100 mM NaCl. No significant effect of salinity on this
parameter was observed in plants inoculated with Sc CdG, while plants inoculated with
Ce CdG enhanced their shoot CAT activity at both 66 and 100 mM NaCl. In root
tissues, CAT activity increased in all treatments after the application of either 66 or 100
mM NaCl. When data were analyzed within each salt level, it was observed that under
non-saline conditions CAT activity was unaffected by inoculation treatments in both
leaves and roots (Fig. 5A, B). At 66 mM NaCl, all the mycorrhizal plants showed higher
CAT activity in their roots than the non-mycorrhizal plants, while in shoots the plants
inoculated with the three native AMF exhibited lower CAT activity than those
inoculated with Ri collect or the non-mycorrhizal plants. Finally, at 100 mM NaCl, no
significant differences in CAT activity were observed in roots, while plants inoculated
with Ri CdG and Ce CdG, had the highest CAT activity in shoots.

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Chapter 5

D
18 a A
16 A
14 W
ab G a
J W
CAT activity (nmol H2O2 min-1mg-1Prot)
12
GH B E a
10 J cd bc J NM
8 B H a X d b b
b Ri collect
6 a F a a Ri CdG
4 a Sc CdG
2 Ce CdG
0
0 mM 66 mM 100 mM

18 D
a G W B
16
14 a a
G J A
J
12 a a a
E X a
10
b b
8
B
6 B
F H K Y c
4 a
a a a a
2
0
0 mM 66 mM 100 mM

Fig. 5. Shoot (A) and root (B) CAT activity in maize plants. See legend for Fig. 1.

4. Discussion

Excessive soil salinity is well known to induce oxidative stress in plants [54].
Previous studies have suggested that tolerance of plants to salt stress is associated with
the induction of antioxidant enzymes and reduction of oxidative damage [55-56]. AM
symbiosis enhances the activity of antioxidant enzymes in order to help plants to cope
with the reactive oxygen species generated by salinity [22-23, 57]. Nevertheless the
response of the individual enzymes varies with respect to the host plant and the fungal
species involved in the association [11]. Results from the present work confirms that
native AMF reduced the oxidative damage in the host plant, but also that they differed
in their response to salinity, in the modulation of the antioxidative capacity of the host
plant and in the promotion of plant growth under saline conditions. The degree of root
colonization also varied among fungal species, but the literature reflects that there is no
threshold value of root colonization for enhancement of plant fitness and this depends
on the plant and the fungal species involved [58]. In fact Ri collect had higher
colonization rate than the autochthonous AMF but it did not show better symbiotic
efficiency.
In this study, AM plants may improve the net assimilation rates by protecting
photochemical processes of photosystem II and enhancing stomatal conductance when
subjected to salinity stress. Plants inoculated with native AMF exhibited better
performance of photosystem II and higher stomatal conductance when subjected to high

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salinity level (100 mM NaCl). These results indicate less damage in the photosynthetic
machinery and enhanced transpiration rates in maize plants inoculated with native
AMF. Sheng et al. [59] and Hajiboland et al. [60] reported a similar tendency, and
Querejeta et al. [27] showed that the enhancement of plant stomatal conductance was
higher with native AMF than with collection fungi. These two effects may have
accounted for the enhanced tolerance of AM plants, particularly native inoculated ones,
most likely by enhancing CO2 fixation and plant growth during salinity stress. In any
case, the native Sc CdG also improved these physiological parameters but it did not
enhance plant growth, demonstrating a differential ability by each fungus or that an
extended growing period was needed to achieve a positive effect on plant growth.
The above mentioned processes could have also contributed to decrease
photorespiration and then lead to lower ROS production in AM plants [10]. Indeed, the
accumulation of hydrogen peroxide under salinity was considerably lower in roots of
plants inoculated with the three native AMF and the oxidative damage to lipids was also
lower in shoots and roots of these AM plants. Several studies have reported lower H2O2
accumulation in AM plants [60-61]. Our results demonstrate that colonization of maize
plants by salinity-adapted AMF lead to a lower accumulation of ROS species. Salt
stress injuries the membrane integrity, which produces ion leakage. Thus one of the key
processes in salinity tolerance is to prevent lipid peroxidation to maintain the membrane
integrity [57, 62]. The oxidation of membrane lipids gives a reliable indication of an
extra free radical production leading to oxidative stress [63]. Data from this study
showed a lower MDA content in native-AMF inoculated plants. A similar reduction of
oxidative damage to lipids by AM symbiosis has been observed in tomato plants
subjected to salinity stress [23, 60]. These data, together with the lower electrolyte
leakage in maize plants inoculated with native AMF from Cabo de Gata, provides
evidence that the presence of native-AM symbiosis can prevent cell membrane injury.
This is in accordance to some other works that showed lower lipid peroxidation and
membrane permeability in AM plants compared to non-mycorrhizal plants [23, 57, 62,
64]. For instance, Kaya et al. [65] also reported that the electrolyte leakage in leaves of
Capsicum annum treated with 50 mM and 100 mM NaCl were decreased significantly
by AM inoculation. Again, plants inoculated with native AMF Ri CdG and Sc CdG had
lower electrolyte leakage than non-mycorrhizal plants or plants inoculated with Ri
collect. Several works have shown that AMF isolated from a saline area exhibited
significant higher symbiotic efficiency than non-saline AMF [19, 29]. Moreover,
Querejeta et al. [27] suggested that modulation of leaf gas exchange parameters in
Mediterranean environments by native-adapted AMF is of critical importance for host
plant performance in semiarid environments.
As discussed previously, the lower MDA concentrations in AM-inoculated
plants may have been due to the better performance of photosystem II and lower
generation of ROS in these plants. However, the activation of antioxidant enzymes such
as superoxide dismutase (SOD) or catalase (CAT) may have also accounted [14, 66].

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Our results showed that AM symbiosis significantly influenced SOD and CAT activities
to different extent depending on the AMF species, which might be the result of a
complex interaction among AMF, maize and the salinity stress. The reactive oxyradical
scavenging enzyme is an antioxidant system that plays a role maintaining cell
membrane stability in plant cell [67]. SOD enzyme catalyses the conversion of free O2•−
to O2 and H2O2. In the present work, the effect of salt stress on antioxidant enzyme
activity in maize revealed that the increase of SOD activity was higher in roots than in
shoots. In the root tissues, at 100 mM NaCl, all native AM-inoculated plants showed
significantly higher SOD activity and Ce CdG had the highest value. Several studies
reported that AMF enhance SOD activity in mycorrhizal plants under salt stress
conditions [57, 60, 68]. The greater SOD activity that we observed in mycorrhizal
plants could increase the capacity to scavenge superoxide radicals. Enhancement of
SOD activity under salt stress after the inoculation with salinity adapted fungi suggests
that native AMF improve the capacity of maize plants to cope with oxidative stress. In a
previous work, we provided evidence that the encoding gene for SOD in the fungus Ri
CdG was up-regulated under saline conditions. Hence we confirmed a fungal response
to the oxidative stress induced by salinity [29] which may have accounted also for the
reduced oxidative damage in the inoculated maize plants. Once the free O2•− is
detoxified by SOD activity, there is a need to scavenge hydrogen peroxide which is still
toxic and must be eliminated by conversion to H2O in subsequent reactions. A number
of enzymes regulate H2O2 intracellular levels in plants, and CAT is among the most
important ones [13]. Several authors have reported an enhancement of CAT activity by
inoculation with AMF [57, 60, 69]. However, in our work the effect of AMF on CAT
activity under salinity stress was little evident, which is in agreement with other reports
indicating that AM symbiosis did not affect CAT activity of salt stressed plants [23, 68].
The effects of AM symbiosis on the antioxidant systems observed in this study
were more prominent under the highest salt stress level (100 mM NaCl). The latter
suggests that under mild salts in the soil, maize antioxidant system could be enough to
cope with the stress. However, at higher levels of salt in the soil, AMF, particularly
salinity-adapted ones, are important to improve the capacity of maize to grow under
such unfavourable conditions. Greater SOD activity in plants inoculated with native
AMF compared with non-mycorrhizal or Ri collect plants was associated with lower
accumulation of H2O2 and less lipid peroxidation, indicating lower oxidative and
membrane damage in the native AMF-inoculated plants. Our observations are in
agreement with Bartels [70] that proposed both the prevention of oxidative stress and
the elimination of ROS as the most effective approaches used by plants to gain
tolerance against several abiotic stresses, including salinity.
Our results supports that AM inoculation enhances maize salt tolerance by
alleviating the salt induced oxidative stress and membrane damage. To this effect, the
better performance of photosystem II and stomatal conductance of plants inoculated
with the three native AMF must also have contributed through reduced ROS generation.

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This work demonstrates that AMF isolated from saline areas appear to be
physiologically adapted to the stress conditions, conferring higher salinity tolerance and
improving the growth of an important crop plant such as maize under salinity stress. In
agricultural lands with high levels of disturbance, inoculation is beneficial due to low
AM fungal inoculum potential [71]. Thus, from this work we highlight the potential use
of AMF from saline habitats to make successful inocula to improve crop growth and
yield in saline soils.

Acknowledgements

This work was financed by two research projects supported by Junta de


Andalucía (Spain). Projects P06-CVI-01876 and P11-CVI-7107. We thank Sonia
Molina for technical assistance and Domingo Álvarez (curator of the EEZ germplasm
collection), for taking care of the native AMF inocula.

References

[1]...IGC, Grain Market Report, in: IGC Grains conference 2012, International Grains
Council, London - 7 June, 2012.
[2]...L.K. Heng, T. Hsiao, S. Evett, T. Howell, P. Steduto, Validating the FAO
AquaCrop Model for irrigated and water deficient field maize, Agron. J., 101
(2009) 488-498.
[3]...K.A. Schwabe, K. Iddo, K.C. Knap, Drain water management for salinity
mitigation in irrigated agriculture, Am. J. Agric. Econ., 88 (2006) 133-140.
[4]...M. Pitman, A. Läuchli, Global impact of salinity and agricultural ecosystems, in:
A. Läuchli, U. Lüttge (Eds.) Salinity: environment-plants-molecules, Kluwer
Academic Publishers, Netherlands, 2002, pp. 3-20.
[5]...S. Mahajan, N. Tuteja, Cold, salinity and drought stresses: an overview, Arch.
Biochem. Biophys., 444 (2005) 139-158.
[6]...R. Munns, M. Tester, Mechanisms of salinity tolerance, Annu. Rev. Plant Biol., 59
(2008) 651-681.
[7]...D. Mejía, Maize: Post-Harvest Operations, AGST-FAO, Rome, 2003.
[8]...T.J. Flowers, Improving crop salt tolerance, J. Exp. Bot., 55 (2004) 307-319.
[9]...A.D. Azevedo-Neto, J.T. Prisco, J. Eneas-Filho, C.E.B. Abreu, E. Gomes-Filho,
Effect of salt stress on antioxidative enzymes and lipid peroxidation in leaves and
roots of salt-tolerant and salt-sensitive maize genotypes, Environ. Exp. Bot., 56
(2006) 87-94.

170
Chapter 5

[10]..S.S. Gill, N. Tuteja, Reactive oxygen species and antioxidant machinery in abiotic
stress tolerance in crop plants, Plant Physiol. Biochem., 48 (2010) 909-930.
[11]..J.M. Ruiz-Lozano, R. Porcel, R. Azcón, R. Aroca, Regulation by arbuscular
mycorrhizae of the integrated physiological response to salinity in plants: new
challenges in physiological and molecular studies, J. Exp. Bot., 63 (2012) 4033-
4044.
[12]..C. Triantaphylides, M. Havaux, Singlet oxygen in plants: production,
detoxification and signaling, Trends Plant Sci., 14 (2009) 219-228.
[13]..G. Miller, N. Suzuki, S. Ciftci-Yilmaz, R. Mittler, Reactive oxygen species
homeostasis and signalling during drought and salinity stresses, Plant Cell
Environ., 33 (2010) 453-467.
[14]..R. Mittler, Oxidative stress, antioxidants and stress tolerance, Trends Plant Sci., 7
(2002) 405-410.
[15]..I. Türkan, T. Demiral, Recent developments in understanding salinity tolerance,
Environ. Exp. Bot., 67 (2009) 2-9.
[16]..J.M. Barea, M.J. Pozo, J.M. López-Ráez, R. Aroca, J.M. Ruíz-Lozano, N. Ferrol,
R. Azcón, C. Azcón-Aguilar, Arbuscular Mycorrhizas and their significance in
promoting soil-plant systems sustainability against environmental stresses in: B.
Rodelas, J. Gonzalez-Lopez (Eds.) Beneficial Plant-Microbial Interactions:
Ecology and Applications Science Publishers, USA, 2012, (In press).
[17]..D. Redecker, R. Kodner, L.E. Graham, Glomalean fungi from the Ordovician,
Science, 289 (2000) 1920-1921.
[18]..S.E. Smith, D.J. Read, Mycorrhizal Symbiosis, 3rd Ed., Elsevier, Academic
Press, New York, 2008.
[19]..M. Yamato, S. Ikeda, K. Iwase, Community of arbuscular mycorrhizal fungi in a
coastal vegetation on Okinawa island and effect of the isolated fungi on growth of
sorghum under salt-treated conditions, Mycorrhiza, 18 (2008) 241-249.
[20]..P. Wilde, A. Manal, M. Stodden, E. Sieverding, U. Hildebrandt, H. Bothe,
Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt
marshes, Environ. Microbiol., 11 (2009) 1548-1561.
[21]..H. Evelin, R. Kapoor, B. Giri, Arbuscular mycorrhizal fungi in alleviation of salt
stress: a review, Ann. Bot., 104 (2009) 1263-1280.
[22]..M.M. Alguacil, J.A. Hernández, F. Caravaca, B. Portillo, A. Roldán, Antioxidant
enzyme activities in shoots from three mycorrhizal shrub species afforested in a
degraded semi-arid soil, Physiol. Plant., 118 (2003) 562-570.
[23]..Z. He, C. He, Z. Zhang, Z. Zou, H. Wang, Changes of antioxidative enzymes and
cell membrane osmosis in tomato colonized by arbuscular mycorrhizae under
NaCl stress, Colloids Surf. B., 59 (2007) 128-133.
[24]..R. Calvente, C. Cano, N. Ferrol, C. Azcón-Aguilar, J.M. Barea, Analysing natural
diversity of arbuscular mycorrhizal fungi in olive tree (Olea europaea L.)
plantations and assessment of the effectiveness of native fungal isolates as

171
Chapter 5

inoculants for commercial cultivars of olive plantlets, Appl. Soil Ecol., 26 (2004)
11-19.
[25]..B. Enkhtuya, J. Rydlova, M. Vosátka, Effectiveness of indigenous and non-
indigenous isolates of arbuscular mycorrhizal fungi in soils from degraded
ecosystems and man-made habitats, Appl. Soil Ecol., 14 (2000) 201-211.
[26]..F. Caravaca, J.M. Barea, J. Palenzuela, D. Figueroa, M.M. Alguacil, A. Roldán,
Establishment of shrubs species in a degraded semiarid site after inoculation with
native or allochthonous arbuscular mycorrhizal fungi, Appl. Soil Ecol., 22 (2003)
103-111.
[27]..J.I. Querejeta, M.F. Allen, F. Caravaca, A. Roldán, Differential modulation of host
plant δ13C and δ18O by native and nonnative arbuscular mycorrhizal fungi in a
semiarid environment, New Phytol., 169 (2006) 379-387.
[28]..R.S. Oliveira, M. Vosátka, J.C. Dodd, P.M.L. Castro, Studies on the diversity of
arbuscular mycorrhizal fungi and the efficacy of two native isolates in a highly
alkaline anthropogenic sediment, Mycorrhiza, 16 (2005) 23-31.
[29]..B. Estrada, J.M. Barea, R. Aroca, J.M. Ruiz-Lozano, A native Glomus
intraradices strain from a Mediterranean saline area exhibits salt tolerance and
enhanced symbiotic efficiency with maize plants under salt stress conditions, Plant
and Soil (In press), (2012) doi: 10.1007/s11104-012-1409-y
[30]..E. Sieverding, Vesicular-arbuscular mycorrhiza management in tropical
agrosystems, TZ-Verlagsgesellschaft, Technical Cooperation (GTZ), Eschborn,
Friedland, Bremer, Rossdorf, Germany, 1991.
[31]..R.E. Koske, B. Tessier, A convenient, permanent slide mounting medium, Myc.
Soc. Am. Newsl., 34 (1983) 59.
[32]..M. Brundrett, L. Melville, L. Peterson, Practical methods in mycorrhizal research,
Mycologue Publications, University of Guelph, Ontario, Canada, 1994.
[33]..J.L. Spain, Arguments for diagnoses based on unaltered wall structures,
Mycotaxon, 38 (1990) 71-76.
[34]..F. Oehl, E. Sieverding, J. Palenzuela, K. Ineichen, G.A. Silva, Advances in
Glomeromycota taxonomy and classification, IMA Fungus, 2 (2011) 191-199.
[35]..N.C. Schenk, G.S. Smith, Additional new and unreported species of mycorrhizal
fungi (Endogonaceae) from Florida, Mycologia, 74 (1982) 77-92.
[36]..W.N. Becker, J.W. Gerdemann, Glomus etunicatus sp. nov., Mycotaxon, 6 (1977)
29-32.
[37]..J.M. Trappe, Three new Endogonaceae: Glomus constrictus, Sclerocystis
clavispora and Acaulospora scrobiculata, Mycotaxon, 6 (1977) 359-366.
[38]..N. Ferrol, R. Calvente, C. Cano, J.M. Barea, C. Azcón-Aguilar, Analysing
arbuscular mycorrhizal fungal diversity in shrub-associated resource islands from
a desertification-threatened semiarid Mediterranean ecosystem, Appl. Soil Ecol.,
25 (2004) 123-133.

172
Chapter 5

[39]..J. Lee, S. Lee, J.P.W. Young, Improved PCR primers for the detection and
identification of arbuscular mycorrhizal fungi, FEMS Microbiol. Ecol., 65 (2008)
339-349.
[40]..S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local
alignment search tool, J. Mol. Biol., 215 (1990) 403-410.
[41]..M. Krüger, C. Krüger, C. Walker, H. Stockinger, A. Schussler, Phylogenetic
reference data for systematics and phylotaxonomy of arbuscular mycorrhizal fungi
from phylum to species level, New Phytol., 193 (2012) 970-984.
[42]..J.M. Phillips, D.S. Hayman, Improved procedure of clearing roots and staining
parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of
infection, Trans. Br. Mycol. Soc., 55 (1970) 159-161.
[43]..M. Giovannetti, B. Mosse, Evaluation of techniques for measuring vesicular
arbuscular mycorrhizal infection in roots, New Phytol., 84 (1980) 489-500.
[44]..K. Oxborough, N.R. Baker, Resolving chlorophyll a fluorescence images of
photosynthetic efficiency into photochemical and non-photochemical components
- calculation of qP and Fv'/Fm' without measuring Fo', Photosynth. Res., 54
(1997) 135-142.
[45]..P.E. Verslues, M. Agarwal, S. Katiyar-Agarwal, J. Zhu, J.K. Zhu, Methods and
concepts in quantifying resistance to drought, salt and freezing, abiotic stresses
that affect plant water status, Plant J., 45 (2006) 523-539.
[46]..G. Minotti, S.D. Aust, The requirement for iron (III) in the initiation of lipid-
peroxidation by iron(II) and hydrogen-peroxide, J. Biol. Chem., 262 (1987) 1098-
1104.
[47]..B. Halliwell, J.M.C. Gutteridge, Free radicals in biology and medicine, Clanderon
Press, Oxford, UK, 1989.
[48]..B.D. Patterson, E.A. Macrae, I.B. Ferguson, Estimation of hydrogen-peroxide in
plant-extracts using titanium (IV), Anal. Biochem., 139 (1984) 487-492.
[49]..R. Aroca, J.J. Irigoyen, M. Sánchez-Díaz, Drought enhances maize chilling
tolerance. II. Photosynthetic traits and protective mechanisms against oxidative
stress, Physiol. Plant., 117 (2003) 540-549.
[50]..R. Aroca, J.J. Irigoyen, M. Sánchez-Díaz, Photosynthetic characteristics and
protective mechanisms against oxidative stress during chilling and subsequent
recovery in two maize varieties differing in chilling sensitivity, Plant Sci., 161
(2001) 719-726.
[51]..M.M. Bradford, A rapid and sensitive method for quantitation of microgram
quantities of protein utilizing principle of protein-dye binding, Anal. Biochem., 72
(1976) 248-254.
[52]..M. Becana, P. Aparicio-Tejo, J.J. Irigoyen, M. Sánchez-Díaz, Some enzymes of
hydrogen peroxide metabolism in leaves and root nodules of Medicago sativa,
Plant Physiol., 82 (1986) 1169-1171.
[53]..H. Aebi, Catalase in vitro, Methods Enzymol., 105 (1984) 121-126.

173
Chapter 5

[54]..R. Santos, T. Franza, M.L. Laporte, C. Sauvage, D. Touati, D. Expert, Essential


role of superoxide dismutase on the pathogenicity of Erwinia chrysanthemi strain
3937, Mol. Plant-Microbe Interact., 14 (2001) 758-767.
[55]..J.A. Hernández, A. Jiménez, P.M. Mullineaux, F. Sevilla, Tolerance of pea (Pisum
sativum L.) to long-term salt stress is associated with induction of antioxidant
defenses, Plant, Cell Environ., 23 (2000) 853-862.
[56]..A.H. Sekmen, I. Türkan, S. Takio, Differential responses of antioxidative enzymes
and lipid peroxidation to salt stress in salt-tolerant Plantago maritima and salt-
sensitive Plantago media, Physiol. Plant., 131 (2007) 399-411.
[57]..N. Garg, G. Manchanda, Role of arbuscular mycorrhizae in the alleviation of
ionic, osmotic and oxidative stresses induced by salinity in Cajanus cajan (L.)
Millsp (pigeonpea), J. Agron. Crop Sci., 195 (2009) 110-123.
[58]..M. Ruíz-Sánchez, R. Aroca, Y. Muñoz, R. Polon, J.M. Ruiz-Lozano, The
arbuscular mycorrhizal symbiosis enhances the photosynthetic efficiency and the
antioxidative response of rice plants subjected to drought stress, J. Plant Physiol.,
167 (2010) 862-869.
[59]..M. Sheng, M. Tang, H. Chen, B. Yang, F. Zhang, Y. Huang, Influence of
arbuscular mycorrhizae on photosynthesis and water status of maize plants under
salt stress, Mycorrhiza, 18 (2008) 287-296.
[60]..R. Hajiboland, N. Aliasgharzadeh, S.F. Laiegh, C. Poschenrieder, Colonization
with arbuscular mycorrhizal fungi improves salinity tolerance of tomato (Solanum
lycopersicum L.) plants, Plant Soil, 331 (2010) 313-327.
[61]..Q.S. Wu, Y.N. Zou, X.H. He, Contributions of arbuscular mycorrhizal fungi to
growth, photosynthesis, root morphology and ionic balance of citrus seedlings
under salt stress, Acta Physiol. Plant., 32 (2010) 297-304.
[62]..G. Feng, F.S. Zhang, X.L. Li, C.Y. Tian, C. Tang, Z. Rengel, Improved tolerance
of maize plants to salt stress by arbuscular mycorrhiza is related to higher
accumulation of soluble sugars in roots, Mycorrhiza, 12 (2002) 185-190.
[63]..G. Noctor, C.H. Foyer, Ascorbate and glutathione: Keeping active oxygen under
control, Annu. Rev. Plant Physiol. Plant Mol. Biol., 49 (1998) 249-279.
[64]..H. Evelin, B. Giri, R. Kapoor, Contribution of Glomus intraradices inoculation to
nutrient acquisition and mitigation of ionic imbalance in NaCl-stressed Trigonella
foenum-graecum, Mycorrhiza, 22 (2012) 203-217.
[65]..C. Kaya, M. Ashraf, O. Sonmez, S. Aydemir, A.L. Tuna, M.A. Cullu, The
influence of arbuscular mycorrhizal colonization on key growth parameters and
fruit yield of pepper plants grown at high salinity, Scientia Hortic., 121 (2009) 1-
6.
[66]..Q.S. Wu, R.X. Xia, Y.N. Zou, Reactive oxygen metabolism in mycorrhizal and
non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress, J.
Plant Physiol., 163 (2006) 1101-1110.

174
Chapter 5

[67]..Z. Huang, C. He, Z. He, Z. Zou, Z. Zhang, The effects of arbuscular mycorrhizal
fungi on reactive oxyradical scavenging system of tomato under salt tolerance,
Agr. Sci. China, 9 (2010) 1150-1159.
[68]..H. Ghorbanli, M. Ebrahimzadeh, M. Sharifi, Effects of NaCl and mycorrhizal
fungi on antioxidative enzymes in soybean, Biol. Plant., 48 (2004) 575-581.
[69]..M. Borde, M. Dudhane, P. Jite, Growth photosynthetic activity and antioxidant
responses of mycorrhizal and non-mycorrhizal bajra (Pennisetum glaucum) crop
under salinity stress condition, Crop Protect., 30 (2011) 265-271.
[70]..D. Bartels, Targeting detoxification pathways: an efficient approach to obtain
plants with multiple stress tolerance, Trends Plant Sci., 6 (2001) 284-286.
[71]..V.B. Beauchamp, C. Walz, P.B. Shafroth, Salinity tolerance and mycorrhizal
responsiveness of native xeroriparian plants in semi-arid western USA, Appl. Soil
Ecol., 43 (2009) 175-184.

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CAPÍTULO 6
CHAPTER 6

Importance of native arbuscular mycorrhizal inoculation in the


halophyte Asteriscus maritimus for successful establishment and
growth under saline conditions

Submitted to Plant and Soil by Estrada B, Aroca R, Azcón-Aguilar C, Barea J.M


and Ruiz-Lozano J.M.
Capítulo 6

Importancia de la inoculación con hongos nativos formadores de


micorrizas arbusculares en la especie halófita Asteriscus maritimus
para su completo establecimiento y crecimiento bajo condiciones de
salinidad.

Resumen

Antecedentes y objetivos:
Los ecosistemas mediterráneos afectados por salinidad se presentan ante un problema
creciente de degradación. La restauración biológica de los ecosistemas salinos se podría
lograr mediante el uso de especies de plantas halófitas junto con la inoculación de
hongos MA adaptados. Una planta de interés para su uso en la restauración de los
ambientes salinos, Asteriscus maritimus es altamente micotrófica. El objetivo del este
estudio fue evaluar la efectividad de hongos MA nativos y alóctonos en el
establecimiento y crecimiento de la halófita A. maritimus bajo condiciones de salinidad.
Métodos:
Se estudió la efectividad simbióticade cuatro aislados de hongos MA (tres nativos de un
suelo salino y uno alóctono de colección) en A. maritimus sometido a niveles crecientes
de salinidad. Se midieron parámetros fisiológicos de la planta en los que los hongos MA
pueden aliviar el efecto negativo del estrés salino, como la conductancia estomática y la
eficiencia del fotosistema II, acumulación de compuestos antioxidantes, actividad de
enzimas antioxidantes o daño oxidativo a lípidos.
Resultados:
Las plantas de A. maritimus mostraron una gran dependencia micorrícica incluso en
ausencia de estrés salino. Al nivel más alto de salinidad, las plantas inoculadas con
hongos nativos MA tuvieron mayor biomasa aérea, eficiencia del fotosistema II,
conductancia estomática, acumulación de glutatión y actividad de varias enzimas
antioxidantes que plantas inoculadas con el hongo MA de colección. Además, a este
nivel de sal, únicamente 30% de las plantas de A. maritimus inoculadas con el hongo
MA de colección sobrevivieron, mientras que en las plantas colonizadas por los hongos
nativos el porcentaje de supervivencia fue del 100%.
Conclusiones:
El presente estudio muestra la importancia de la inoculación con hongos nativos en
establecimiento, supervivencia y crecimiento de A. maritimus. El uso de un inóculo
adecuado de hongos MA nativos puede ser un elemento crucial para el éxito en la
recuperación de áreas salinas degradadas.

Palabras clave: micorrizas arbusculares nativas, ecosistemas mediterráneos, halófitas,


restauración, salinidad

179
180
Chapter 6

Importance of native arbuscular mycorrhizal inoculation in the


halophyte Asteriscus maritimus for successful establishment and
growth under saline conditions

Beatriz Estrada, Ricardo Aroca, Concepción Azcón-Aguilar, José Miguel Barea


and Juan Manuel Ruiz-Lozano*

Departamento de Microbiología del Suelo y Sistemas Simbióticos. Estación


Experimental del Zaidín (CSIC). Profesor Albareda nº 1, 18008 Granada, Spain.

* Corresponding author: Dr. Juan Manuel Ruiz-Lozano.


e-mail: juanmanuel.ruiz@eez.csic.es
Telph. + 34 958 181600
Fax: + 34 958 129600

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Chapter 6

Abstract

Background and aims:


Salt affected Mediterranean ecosystems have an increasing problem of degradation. The
biological restoration of saline habitats could be achieved by using halophyte plant
species together with adapted arbuscular mycorrhizal fungi (AMF). An interesting plant
to be used in restoration of saline environments, Asteriscus maritimus, is highly
mycotrophic. The aim of this study was to assess the effectiveness of native and
allochthonous AMF to enhance the establishment and growth of the halophyte A.
maritimus under saline conditions.
Methods:
We studied the symbiotic effectiveness of four AMF strains (three native fungal isolates
from a saline soil and one allochthonous, from collection) in A. maritimus subjected to
increasing salinity stress. We measured plant physiological parameters by which AMF
may ameliorate the detrimental effects of salinity stress such as stomatal conductance
and efficiency of photosystem II, accumulation of antioxidant compounds, antioxidant
enzyme activities or oxidative damage to lipids.
Results:
A. maritimus plants showed a high mycorrhizal dependency, even in absence of salt
stress. At the highest level of salinity, plants inoculated with native AMF had higher
shoot dry weight, efficiency of photosystem II, stomatal conductance, accumulation of
glutathione and activity of certain antioxidant enzymes than those inoculated with the
collection AMF. Moreover, at this salt level, only 30% of A. maritimus plants
inoculated with the collection AMF survived, while with the three native AMF, the rate
of survival was 100%.
Conclusions:
Results points out the importance of native AMF inoculation in the establishment,
survival and growth of A. maritimus plants. Thus, the use of adequate native AMF
inocula could be a critical issue for success in recovering saline degraded areas.

Key words: native arbuscular mycorrhiza fungi; Mediterranean ecosystems; halophyte;


restoration; salinity.

Introduction

Mediterranean regions are characterized by very limited rainfall, high light


irradiance and maximum air temperatures largely above 30ºC in summer, which
difficult plant growth (Brito et al. 2011). The latter may provoke the loss of natural
plant communities and accelerate the processes of soil degradation and environmental

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changes (Alguacil et al. 2011). These conditions make Mediterranean ecosystems


fragile and susceptible to degradation and desertification (Ferrol et al. 2004). Thus in
Mediterranean areas, global change will involve not only increased in aridity but also
significant changes in land use (Valladares 2004). This multiple stress situation is
promoting desertification of large areas in Southeast Spain and an alarming increase in
salinity. Salinization of soils is a major problem, not only agronomical but also
ecological, in particular in arid and semiarid ecosystems, where water is scarce and salts
cannot be properly dissolved and they accumulate in the soil (Evelin et al. 2009). High
salinity induces ionic toxicity, osmotic stress and leads to secondary oxidative stress in
plants (Ding et al. 2010). The excess of reactive oxygen species (ROS) production can
damage the structures of enzymes and other macromolecules in plant cells (Mittler
2002). One of the most important tasks to restore the productivity of saline lands is to
improve soil conditions, reduce desertification and raise the fertility of soils (Tawfik et
al. 2010). Hence in restoration of Mediterranean ecosystems there is a need to look for
ecophysiological features that enhance plant performance under conditions that will be
exacerbated by climate change, such as salinity (Vallejo et al. 2005).
The below-ground microbial communities, particularly arbuscular mycorrhiza
fungi (AMF), are well known to improve soil structure and benefit plant performance by
helping in the establishment, enhancing the resistance to environmental stresses and
increasing plant nutrient and water uptake (Jeffries and Barea 2012). Mycorrhizal
symbiosis is universally distributed among the majority of plants and forms a network
of extra-radical mycelium that provides a direct physical link between the plant root and
the soil (Smith and Read 2008). Moreover, they affect the diversity and productivity of
plants helping in the stability and sustainability of ecosystems (van der Heijden et al.
1998). Although is has been reported that excess of salts in the soil inhibits spore
germination and growth of AMF (Juniper and Abbott 2006), several studies found a
high diversity of those fungi in saline soils (Yamato et al. 2008; Wilde et al. 2009).
Even more, in arid and semiarid Mediterranean regions, AMF play a key ecological role
in the functioning of ecosystems (Requena et al. 1996).
On the other hand, halophytes, which constitute about 1% of the world flora, are
specialised plants that can tolerate and complete their life cycles under high levels of
salts in the soil (Munns and Tester 2008). They are physiologically and biochemically
adapted to grow in saline soils (Tawfik et al. 2010). Most of the halophytes in saline
sites belong to the Chenopodiaceae, Juncaceae, Cyperaceae or Brassicaceae families
which are non-or weakly mycotrophic plants. However, associations between AMF and
halophytes are also widely formed, including Aster tripolium, Inula crithmoides,
Plantago maritima, Salsola soda and Suaeda maritima (Evelin et al. 2009; Sonjak et al.
2009). For the present study we selected a native halophyte of lands surrounding the
Mediterranean Sea, especially Spain, Asteriscus maritimus (L.) Less. (Lendínez et al.
2011). Rodríguez et al. (2005) considered this plant as an useful species in revegetation
programmes in Mediterranean areas affected by salinity. A. maritimus is a member of

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the Asteraceae family and known since long time to be highly mycotrophic (Mason
1928). In fact native plants thrive in various abiotically stressed ecosystems thanks to
AMF that have co-evolved and are essential for their adaptation to stressed conditions
(Rodriguez and Redman 2008). Nevertheless, in arid and semiarid ecosystems there is a
low density of AM propagules making difficult the successful reestablishment of native
plants (Jeffries et al. 2002). Thus, it is very important the ecophysiological study of
autochthonous AMF isolates and the knowledge of their mechanisms of salinity stress
adaptation and tolerance (Enkhtuya et al. 2000) in order to select the most adapted and
efficient species/strains of AMF to serve as inocula in revegetation programs (Ferrol et
al. 2004). Both halophytes and indigenous AMF isolates from saline habitats have
developed a variety of modifications to survive in saline environments, such as
regulating ionic homeostasis and detoxifying ROS (Zhu 2001; Ruiz-Lozano et al. 2012).
The biological restoration of saline ecosystems could be achieved by using halophytes
together with inoculation of adapted AMF. However the biochemical mechanisms by
which AM-colonized halophytic plants tolerate or reduce salt stress are poorly
understood.
In the present work, we studied the symbiotic effectiveness of four AMF strains
(three native isolates from a saline soil and one allochthonous, belonging to the EEZ
collection) in Asteriscus maritimus subjected to salinity stress. The native strains of
AMF were successfully isolated from the rizhosphere of A. maritimus at Cabo de Gata
Natural Park (Almería, SE Spain), which is the most arid ecosystem in Europe (Geiger
1973) with important salinity problems as well. The aim was to assess the effectiveness
of AM association among different AM isolates under saline conditions, to enhance the
establishment and growth of the halophyte A. maritimus for revegetation in salt affected
areas. In addition, we tried to elucidate the physiological and biochemical basis of the
mechanisms involved in the variability of the symbiotic performance.

Materials and methods

Identification of the mycorrhizal strains isolated from Cabo de Gata Natural Park

AMF spores were separated from the soil samples by a wet sieving process
(Sieverding 1991). The morphological spore characteristics and their subcellular
structures were described from a specimen mounted in: polyvinyl alcohol-lactic acid-
glycerine (PVLG) (Koske and Tessier 1983); a mixture of PVLG and Melzer’s reagent
(Brundrett et al. 1994); a mixture of lactic acid to water at 1:1; Melzer’s reagent; and
water (Spain 1990). For identification of the AMF species, spores were then examined
using a compound microscope at up to 400-fold magnification as described for
glomeromycotean classification by Oehl et al. (2011). The species were identified based
on its spore morphology as a Rhizophagus intraradices (Schenk and Smith 1982),

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Claroideoglomus etunicatum (Becker and Gerdemann 1977) and Septoglomus


constrictum (Trappe 1977).
In addition to the morphological identification, a molecular identification was
also carried out. For that, spores isolated from the bait cultures of each fungal strain
were surface-sterilized with chloramine T (2%) and streptomycin (0.02%) and crushed
with a sterile disposable micropestle in 40 μL milli-Q water (Ferrol et al. 2004). A two-
step PCR was conducted to amplify the AMF DNA from the spores. The first PCR step
was performed with the universal eukaryote primers NS1 and NS4 region of the small
subunit ribosomal gene and the second with the specific AM fungal primers AML1 and
AML2 (Lee et al. 2008). The amplified DNA was purified using the Ilustra™ GFX™
PCR DNA and Gel Band Purification Kit (GE Helthcare, UK). DNA fragments were
sequenced on an automated DNA sequencer (Perkin-Elmer ABI Prism 373). Sequence
data were compared to gene libraries (EMBL and GenBank) using BLAST program
(Altschul et al. 1990).
The BLAST analysis unambiguously placed Rhizophagus intraradices as the
closest relative of our Rhizophagus intraradices CdG strain, with sequence accession
number FR750209 (Krüger et al. 2012) having a 99% identity. Septoglomus constrictum
was the closest relative to our Se. constrictum CdG strain, with a sequence accession
number FR750212 (Krüger et al. 2012) having a 99% identity. Finally,
Claroideoglomus etunicatum was the closest relative of our Cl. etunicatum CdG strain,
with sequence accession number FR750216.1 (Krüger et al. 2012) having also a 99%
identity. The AM fungal strains have been incorporated to the collection of Zaidin
Experimental Station, Granada, Spain, under accession numbers EEZ 195, EEZ 196 and
EEZ 163, respectively.

Experimental design

The experiment consisted of a randomized complete block design with five


inoculation treatments: (1) non-mycorrhizal control plants, (2) plants inoculated with
the model AM fungus Rh. intraradices (Ri collect) reproduced at the collection of the
Zaidin Experimental Station (isolated EEZ 01), (3) plants inoculated with the AM
fungal strain Rh. intraradices isolated from Cabo de Gata Natural Park (Ri CdG), (4)
plants inoculated with the AM fungal strain Se. constrictum isolated from CdG (Sc
CdG) and (5) plants inoculated with the AM fungal strain Cl. etunicatum isolated from
CdG (Ce CdG). There were 30 replicates of each inoculation treatment, totalling 150
pots (one plant per pot), so that ten of each microbial treatment were grown under
nonsaline conditions throughout the entire experiment (only the salinity provided by the
soil/sand mixture used), while ten pots per treatment were subjected to 100 mM of NaCl
and the remaining ten pots per treatment were subjected to 175 mM of NaCl.

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Soil and biological materials

Loamy soil was collected from Cabo de Gata Natural Park (Almería province)
(Spain, 36º45´24´´N 02º13´17´´W), sieved (5 mm), diluted with quartz-sand (<2 mm)
(1:1, soil:sand, v/v) and sterilized by steaming (100ºC for 1 h on 3 consecutive days).
The original soil had a pH of 8.7 [measured in water 1:5 (w/v)]; 0.26 % organic matter,
nutrient concentrations (g kg-1): N, 0.3; available P, 47.0 and soil electrical conductivity
3.95 dS m-1.
Seeds of Asteriscus maritimus. L were sown on a vermiculite:sand mixture (1:1,
v/v) for germination. Two weeks after germination, four seedlings of A. maritimus were
transplanted per pot containing 900 g of the same soil/sand mixture as described above
and thinned to one plant per pot after establishment was successfully done (plants were
selected with uniform size for each treatment).

Inoculation treatm

Mycorrhizal inoculum consisted of soil, spores, mycelia and infected root


fragments. The AMF species used were three strains isolated from Cabo de Gata
Natural Park (Almería, Spain): Rhizophagus intraradices (previously named Glomus
intraradices), Septoglomus constrictum and Claroideoglomus etunicatum and a
Rhizophagus intraradices strain from our culture collection. Appropriate amounts of
each inoculum containing about 700 infective propagules (according to the most
probable number test), were added to the corresponding pots at transplanting time just
below A. maritimus plantlets. Non-mycorrhizal control plants received the same amount
of autoclaved mycorrhizal inocula together with a 10 ml aliquot of a filtrate (< 20 μm)
of the four AM inocula in order to provide a general microbial population free of AM
propagules.

Growth conditions

The experiment was carried out under glasshouse conditions with temperatures
ranging from 19 to 25ºC, 16/8 light/dark period, and a relative humidity of 50-60%. A
photosynthetic photon flux density of 800 μE m-2 s-1 was measured with a light meter
(LICOR, Lincoln, NE, USA, model LI-188B). Water was supplied daily to the entire
period of plant growth to avoid any drought effect. Plants were established for 6 weeks
prior to salinization to allow adequate plant growth and symbiotic establishment. Three
concentrations (0, 100, and 175 mM NaCl) of saline solution were reached in the soil
substrate by adding appropriate dilutions of a stock 2 M saline solution. The
concentration of NaCl in the soil was increased gradually on alternative days to avoid an
osmotic shock. It took 6 weeks, to reach the desired 100 and 175 mM NaCl levels. The
electrical conductivities in the soil:sand mixture used as growing substrate were 2.2, 8.9

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and 13.5 dS m-1 for the salt levels of 0, 100, and 175 mM NaCl, respectively. Plants
were maintained under these conditions for additional 8 weeks.

Symbiotic development

The percentage of mycorrhizal root infection in A. maritimus plants was


estimated by visual observation of fungal colonization after clearing washed roots in
10% KOH and staining with 0.05% trypan blue in lactic acid (v/v), as described by
Phillips and Hayman (1970). The extent of mycorrhizal colonization was calculated
according to the gridline intersect method Giovannetti and Mosse (1980).

Biomass production

At harvest (5 months after planting), the shoot and root system were separated
and the shoot dry weight (SDW) and root dry weight (RDW) were measured after
drying in a forced hot-air oven at 70ºC for two days. Ten plants per treatment were used
(except for plants inoculated with Ri collect and subjected to 175 mM NaCl, where only
three plants survived).

Photosynthetic efficiency

The efficiency of photosystem II was measured with FluorPen FP100 (Photon


Systems Instruments, Brno, Czech Republic), which allows a non-invasive assessment
of plant photosynthetic performance by measuring chlorophyll a fluorescence. FluorPen
quantifies the quantum yield of photosystem II as the ratio between the actual
fluorescence yield in the light-adapted state (FV‘) and the maximum fluorescence yield
in the light-adapted state (FM‘), according to Oxborough and Baker (1997).
Measurements were taken in the third youngest leaf of ten different plants of each
treatment.

Stomatal conductance

Stomatal conductance was measured two hours after light turned on by using a
porometer system (Porometer AP4, Delta-T Devices Ltd, Cambridge, UK) following
the user manual instructions. Stomatal conductance measurements were taken in the
third youngest leaf from five different plants from each treatment.

Proline content

Free proline was extracted from 0.5 g of fresh leaves and roots (Bligh and Dyer
1959) in five plants per treatment. The methanolic phase was used for quantification of

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Chapter 6

proline content. Proline was estimated by spectrophotometric analysis at 530 nm of the


ninhydrin reaction according to Bates et al. (1973).

Glutathione content

Glutathione content was measured as described by (Smith 1985). Five hundred


milligrams of roots and leaves of five plants from each treatment were homogenized in
a cold mortar with 5 ml 5% (w/v) sulfosalicylic acid and the homogenate was filtered
and centrifuged at 10,000 x g for 10 min. 0.75 ml of supernatant was neutralized by
1.125 ml 0.5 M K-phosphate buffer (pH 7.5). The standard incubation medium was a
mixture of: 0.5 ml 0.1 M sodium phosphate buffer (pH 7.5) containing 5 mM EDTA,
0.2 ml 6 mM 5,5´- dithiobis-(2-nitrobenzoic acid), 0.1 ml 2 mM NADPH, and 0.1 ml (1
unit) glutathione reductase. The reaction was initiated by the addition of 0.1 ml of
extract or glutathione. The change in absorbance at 412 nm was recorded for 9 min.

Ascorbate content

Ascorbate was assayed photometrically by the reduction of 2,6-


dichlorophenolindophenol (DCPIP) as described by Leipner et al. (1997). Two hundred
mg of roots and leaves of five plants from each treatment were homogenized in 5 ml
ice-cold 2% (w/v) metaphosphoric acid in the presence of 1 g NaCl. The homogenate
was filtered through a filter paper. An aliquot of 30 μl of the extract was mixed with 20
μl 45% (w/v) K2HPO4. After 15 min incubation at 25ºC, 100 μl 2 M citrate-phosphate
buffer (pH 2.3) and 100 μl 0.003% (w/v) DCPIP were added. The absorbance at 524nm
was measured immediately. The content of ascorbate was calculated by reference to a
standard curve made of ascorbate.

Oxidative damage to lipids

Lipid peroxides were extracted by grinding 500 mg of leaves and roots of five
plants from each treatment with an ice-cold mortar and 6 ml of 100 mM potassium
phosphate buffer (pH 7). Homogenates were filtered through one Miracloth layer and
centrifuged at 15,000 × g for 20 min. The chromogen was formed by mixing 200 μl of
supernatants with 1 ml of a reaction mixture containing 15% (w/v), trichloroacetic acid,
0.375% (w/v) 2-thiobarbituric acid (TBA), 0.1% (w/v) butyl hydroxytoluene, 0.25N
HCl and then incubating the mixture at 100ºC for 30 min (Minotti and Aust 1987). After
cooling at room temperature, tubes were centrifuged at 800 × g for 5 min and the
supernatant was used for spectrophotometric reading at 532 nm. Lipid peroxidation was
estimated as the content of 2-thiobarbituric acid-reactive substances (TBARS) and
expressed as equivalents of malondialdehyde (MDA) according to Halliwell and
Gutteridge (1989). The calibration curve was made using MDA in the range of 0.1-10

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Chapter 6

nmol. A blank for all samples was prepared by replacing the sample with extraction
medium, and controls for each sample were prepared by replacing TBA with 0.25N
HCl. In all cases, 0.1% (w/v) butyl hydroxytoluene was included in the reaction
mixtures to prevent artifactual formation of TBARS during the acid-heating step of the
assay.

Antioxidant enzyme activities

Enzyme extraction was done as described before by Aroca et al. (2001) with
slight modifications. Five hundred mg of leaf and root fresh tissues were homogenized
separately in a cold mortar with 4 ml of 100 mM phosphate buffer (pH 7.0) containing
0.1 mM DTPA (diethylenetriamine pentaacetic acid; a metal chelating agent) and 40 mg
PVPP (polyvinylpolypyrrolidone), which removes phenolics and alkaloids from plant
extracts, avoiding interference with spectrophotometric measurements and enhancing
enzyme stability. The homogenate was centrifuged at 20,000 x g for 20 min at 4ºC. The
supernatant was separated and used to determine the activity of antioxidant enzymes.
All enzymatic activities were measured in a spectrophotometer InfiniteR 200 PRO series
(Tecan Trading AG, Switzerland) at 25ºC. Total protein was assayed by the method
described by Bradford (1976).

Superoxide dismutase (SOD.) (EC 1.15.1.1) activity was measured according to


Becana et al. (1986). The reaction mixture (1,9 ml) contained 50 mM phosphate buffer
(pH 7.8), 14,3 μM methionine, 82,5 μM NBT, 2,2 μM riboflavin and 100 μl enzyme
extract. Riboflavin was added last and tubes were shaken and illuminated with
fluorescent light. The reaction was allowed to proceed for 10 min until the colour of the
blank shifted to dark violet. Absorbance of the reaction mixture was read at 560 nm.
One unit of SOD activity (U) was defined as the amount of enzyme which produced a
50% inhibition of nitroblue tetrazolium (NBT) reduction.

Glutathione reductase (GR) (EC 1.6.4.2), was assayed as described by Schaedle


and Bassham (1977) with slight modifications as 200 μl reaction mixture containing 50
mM Tris-HCl (pH 7.5), 0.3 mM NADPH2, 1 mM oxidized glutathione (GSSG), and 3
mM MgCl2. The oxidation of NADPH2 was recorded at 340 nm for 3 min after adding
10 μl of leaf and root extract.

Ascorbate peroxidase (APX) (EC 1.11.1.11) was assayed as described by


Nakano and Asada (1981) with slight modifications as a 200 μl reaction mixture
containing 80 mM phosphate (pH 7), 1 mM ascorbate, and 0.5 mM H2O2. The oxidation
of ascorbate was recorded at 290 nm for 3 min after adding 10 μl of leaf and root
extract.

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Chapter 6

Statistical Analysis

Statistical analysis was performed using SPSS 19.0 statistical program (SPSS
Inc., Chicago, IL, USA) performing first a one-way ANOVA followed by the Tukey
test with P< 0.05 as the significance cut-off. Two independent statistical analyses were
carried out: the first to analyze data from the different AMF treatments within each
saline level and the second one to analyze data from each fungal species at increasing
salinity.

Results

Symbiotic development

All the fungi exhibited similar colonization rates at all salinity levels (Table 1).
Within each salinity level, the highest rate of AM root colonization was achieved in
plants inoculated with Ri collect (up to 77%). High levels of root colonization were also
found in plants inoculated with Ri CdG and Sc CdG (Table 1). In contrast, the lowest
root colonization was always found in plants colonized by Ce CdG (about 25%).

Percentage of survival and plant biomass production

Non-mycorrhizal A. maritimus plants did not survive under the conditions of this
experiment, even in the absence of salinity. At the highest salinity level, only 30% of
plants inoculated with Ri collect survived. The rest of the inoculation treatments had
100 % of survival rate (Table 1).
The increase of salt application affected negatively the shoot biomass production
in all treatments except in plants inoculated with Ri CdG (Table 1). In plants inoculated
with Sc CdG and Ce CdG the decrease was only observed at 100 mM. In plant
inoculated with Ri collect the reduction was not significant until 175 mM NaCl was
reached (Table 1). However, under the highest saline conditions, shoot dry weight was
higher in plants colonized by AMF from Cabo de Gata than in those colonized by Ri
collect (Table 1). In contrast, the root biomass production decreased at each salinity
level in plants inoculated with Ri both collect and CdG, two of them showing the lowest
values of root dry weight at 175 mM NaCl (Table 1). Root dry weight was unaffected in
plants inoculated with Ce CdG. Root dry weight was reduced to a half in plants
inoculated with Sc CdG at 100 mM NaCl and it was not changed further at 175 mM
NaCl, being the highest value at this salt level (Table 1).

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Chapter 6

Table 1. Percentage of mycorrhizal root colonization, percentage of survival, shoot and root dry weight
(g plant-1) in Asteriscus maritimus plants. NM represents non-mycorrhizal control plants; Ri collect,
plants inoculated with the collection Rhizophagus intraradices strain; Ri CdG, plants inoculated with the
native Rh. intraradices CdG strain; Sc CdG, plants inoculated with the native Septoglomus claroideum
CdG strain and Ce CdG plants inoculated with the native Claroideoglomus etunicatum CdG strain. Plants
were subjected to 0, 100 or 175 mM NaCl. Different letters indicate significant differences (p < 0.05)
among fungal treatments at each salt level (a, b, c, d) or among salt levels for each AMF treatment: Ri
collect (A, B, C), Ri CdG (D, E, F), Sc CdG (G, H, I) or Ce CdG (J, K, L).

AMF NaCl AM root % Shoot dry Root dry


treatment (mM) colonization (%) survival weight weight

0 0 0 0 0
NM 100 0 0 0 0
175 0 0 0 0

0 82.0 ± 1.7 Aa 100 0.41 ± 0.040 Abc 0.37 ± 0.041 Ab


Ri collect 100 77.7 ± 2.2 Aa 100 0.36 ± 0.032 Aa 0.21 ± 0.035 Bb
175 81.0 ± 1.5 Aa 30 0.22 ± 0.007 Bb 0.11 ± 0.004 Cc

0 55.3 ± 2.4 Db 100 0.36 ± 0.022 Dc 0.43 ± 0.052 Db


Ri CdG 100 61.7 ± 2.3 Db 100 0.33 ± 0.048 Da 0.28 ± 0.033 Eab
175 56.7 ± 2.5 Db 100 0.31 ± 0.041 Da 0.15 ± 0.026 Fbc

0 46.0 ± 3.8 Gb 100 0.58 ± 0.041 Ga 0.61 ± 0.057 Ga


Sc CdG 100 51.7 ± 2.3 Gc 100 0.42 ± 0.025 Ha 0.35 ± 0.043 Ha
175 47.3 ± 3.0 Gc 100 0.33 ± 0.054 Ha 0.28 ± 0.035 Ha

0 24.7 ± 2.3 Jc 100 0.50 ± 0.026 Jab 0.33 ± 0.028 Jb


Ce CdG 100 27.3 ± 1.8 Jb 100 0.39 ± 0.031 Ka 0.23 ± 0.025 Jb
175 28.7 ± 1.5 Jb 100 0.32 ± 0.030 Ka 0.24 ± 0.046 Jab

Photosystem II efficiency
Salinity application did not cause any significant descent in photosynthetic
efficiency in A. maritimus plants inoculated with the three native AMF from Cabo de
Gata (Fig. 1A). Only plants inoculated with Ri collect decreased the efficiency of
photosystem II when they were subjected to 175 mM NaCl (Fig. 1A).

Stomatal conductance

Under non saline conditions no significant differences in stomatal conductance


were observed among inoculation treatments (Fig. 1B). Salinity application decreased
stomatal conductance in all the treatments. However, at 175 mM NaCl, plants
inoculated with Ri collect showed lower stomatal conductance than the three native
AMF from Cabo de Gata (Fig. 1B).

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Chapter 6

0.7 A D G J A

Photosynthetic efficiency
G D
a D a J G J
a a a a a A
a a a a
0.6

0.5 B
b
0.4

0.3
0 mM 100 mM 175 mM

180 Ri collect
J
Ri CdG B
160 G a Sc CdG
D
Stomatal conductance (mmolH2Om-2s-1)

A a Ce CdG
140 a
a
120

100
80

H
25 B E H K K
E a
a a a a B a
a
20
b
15
10
5
0
0 mM 100 mM 175 mM

Fig. 1. Efficiency of photosystem II (A) and stomatal conductance (mmol H2O m-2s-1) (B) in Asteriscus
maritimus plants. Black bars represent plants inoculated with the collection Rhizophagus intraradices
strain (Ri collect); white bars, plants inoculated with the native Rh. intraradices CdG strain (Ri CdG);
grey bars, plants inoculated with the native Septoglomus claroideum CdG strain (Sc CdG) and lined bars,
plants inoculated with the native Claroideoglomus etunicatum CdG strain (Ce CdG). Plants were
subjected to 0, 100 or 175 mM NaCl. Different letters indicate significant differences (p < 0.05) among
fungal treatments at each salt level (a, b, c) or among salt levels for each AMF treatment: Ri collect plants
(A, B, C), Ri CdG (D, E, F), Sc CdG (G, H, I) or Ce CdG (J, K, L).

Accumulation of proline

The accumulation of proline was more pronounced in shoot than in root tissues
(Fig. 2A,B). In leaves proline increased after exposure to salinity in the growth medium.
Differences between treatments were only evident at 100 mM NaCl, where plants
inoculated by any of the two Ri strains had the highest values (Fig. 2A). In roots only
plants inoculated with Ri CdG increased the accumulation of proline with increasing
salinity. All the other fungi increased the accumulation of proline at 100 mM and did
not change it at 175 mM NaCl (Fig. 2B). A. maritimus inoculated with Ri CdG or Ce
CdG had the lowest proline value at 100 mM NaCl(Fig. 2B).

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Chapter 6

20 A D G J
B
E a a a a A
a
a
15
K
H
b
10 b
Proline content (μmol g-1 DW)

Ri collect
5 I L Ri CdG
C F Sc CdG
a a a a Ce CdG
0
0 mM 100 mM 175 mM

A D
A
G G J
14 a a
a E
ab
J a a B
12 b
b
10
8
6
4
B F H K
2 a a a a
0
0 mM 100 mM 175 mM

Fig. 2. Shoot (A) and root (B) proline content in A. maritimus plants. See legend for Fig. 1.

Glutathione content

The highest level of salinity increased the content of glutathione in leaves of


plants inoculated with the three native AMF from Cabo de Gata (Fig. 3A). In contrast,
A. maritimus inoculated with Ri collect reduced the accumulation of glutathione at 175
mM NaCl, having the lowest content. In roots, only plants inoculated with Sc CdG
increased the content of glutathione at 175 mM NaCl, showing the highest content (Fig.
3B).

Ascorbate content

In leaves, the content of ascorbate due to the increasing salinity only rose in Ri
CdG-inoculated plants at 100 mM NaCl and in plants colonized by Ri collect when
subjected to 175 mM NaCl (Fig. 4A). At 100 mM NaCl plant inoculated with Ce CdG
showed the lowest ascorbate content but at the higher level of NaCl no differences were
observed. In roots, exposure to 100 or 175 mM NaCl did not significantly affect the
ascorbate content in A. maritimus plants (Fig. 4B). But again, at 100 mM NaCl
treatment, Sc CdG roots showed the highest ascorbate values. At 175 mM NaCl the
highest values were found in Ri collect roots instead.

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Chapter 6

120 J
D G a A
100 A GH
A D a a
a E ab K
80
K ab a
Glutathione content (nmol g-1 DW)

60 a b B
H ab b Ri collect
40 b Ri CdG
20 Sc CdG
Ce CdG
0
0 mM 100 mM 175 mM

G
80 a
A D D B
60 H H J
D a a AB b
B a a J ab J
b b
40 a a b
20

0
0 mM 100 mM 175 mM

Fig. 3. Shoot (A) and root (B) glutathione content in A. maritimus plants. See legend for Fig. 1.

G G
16 a D A G
a
J ab a J A
14 B B DE a
12 ab a
ab E bc J
a
10 b
c
8
Ascorbate content (mmol g-1 DW)

6 Ri collect
4 Ri CdG
Sc CdG
2 Ce CdG
0
0 mM 100 mM 175 mM

A
10 A J G a
a D a B
a
8 D GH ab D J
Ri collect
a a B J b H ab Ri CdG
6 b b Sc CdG
b
Ce CdG
4

0
0 mM 100 mM 175 mM

Fig. 4. Shoot (A) and root (B) ascorbate content in A. maritimus plants. See legend for Fig. 1.

194
Chapter 6

Oxidative damage to lipids

The application of 100 mM NaCl increased lipid peroxidation in leaves of all


plants except those inoculated with Sc CdG (Fig. 5A). When plants were grown at 175
mM NaCl, only inoculation with Sc CdG enhanced lipid peroxidation as compared to
100 mM NaCl. However these plants showed the lowest values of lipid peroxidation in
their leaves as compared to the other treatments. In roots, salinity application did not
cause any significant increase in lipid peroxidation in plants inoculated with Ri collect,
Ri CdG or Ce CdG (Fig. 5B). Plants inoculated with Sc CdG increased root lipid
peroxidation with increasing salinity. However, the values were always lower than those
of the remaining treatments (Fig. 5B).

A A
30 a D a D
a J A
25 E a
J
Oxidative damage to lipids (nmol MDA g-1 DW)

a
20 a b
B
K G
15 ab
H bc b
H
10 c Ri collect
c Ri CdG
5 Sc CdG
Ce CdG
0
0 mM 100 mM 175 mM

30 A
a A D A B
25 a
D a ab D J
20 J a
b J a
b
15 b G
10 H b
I c
5
c
0
0 mM 100 mM 175 mM

Fig. 5. Shoot (A) and root (B) oxidative damage to lipids in A. maritimus plants. See legend for Fig. 1.

Antioxidant enzyme activities

Leaf SOD activity increased considerably by both salt treatments in plants


inoculated with Sc CdG and Ce CdG (Fig. 6A). Plants inoculated with Ri CdG did not
alter significantly their leaf SOD activity by any of the salt treatments and those
inoculated with Ri collect reduced the enzyme activity at 100 mM NaCl (Fig. 6A).
When data were analyzed within each salt level, plants colonized by Ce CdG showed

195
Chapter 6

the lowest activity at 0 and 100 mM NaCl, together with Ri collect-inoculated plants
(Fig. 6A). At 175 mM NaCl plants inoculated with Sc CdG had the highest SOD
activity, followed by those inoculated with Ce CdG. Root SOD activity increased by
salt application in plants colonized by Sc CdG (Fig. 6B). Ri CdG inoculated plants
showed the lowest SOD activity under non-saline conditions. No significant differences
in root SOD activity among inoculation treatments were observed at 100 mM NaCl
(Fig. 6B).

G
6 Ri collect a
Ri CdG A
5 Sc CdG J
Ce CdG H
4 b
a
D
3 A H D
SOD activity (U min-1mg-1prot)

a
a D a c
2
a B
K K
1 b
b b
nd
0
0 mM 100 mM 175 mM

G
20
a
B
15
A
A H J
10 a D H J
a a a D J
a a a
E b b
5
b
nd

0
0 mM 100 mM 175 mM

Fig. 6. Shoot (A) and root (B) SOD activity in A. maritimus plants. See legend for Fig. 1. (nd means non-
determined enzyme activity).

Under non-saline conditions, no differences in leaf GR activity were observed


among fungal treatments. The GR activity in leaves was only increased at 100 mM
NaCl in plants inoculated with Sc CdG, reaching the highest value. However it did not
increase further at 175 mM NaCl (Fig. 7A). A. maririmus plants inoculated with Ri CdG
and Ce CdG rose their leaf GR activity at 175 mM NaCl, with Ri CdG-inoculated plants
having the highest GR activity (Fig. 7A). Plants inoculated with Ri collect did not rise
their GR activity. In roots, plants inoculated with Sc CdG enhanced their GR activity
with increasing salinity, and those inoculated with Ri CdG also did it at 175 mM NaCl
(Fig. 7B).

196
Chapter 6

D
40 Ri collect a
35 Ri CdG G A
Sc CdG a G J
30 Ce CdG
b b
25 H K
GR activity (nmol NADPH min-1mg-1prot) K
20 A E a a A E
b
15 a a b b
10
5

nd
0
0 mM 100 mM 175 mM

70 G
a B
60
50 H
D
40 a
I b
30 A
A E a J
b E JK
20 ab ab K b c
b
10 b
nd

0
0 mM 100 mM 175 mM

Fig. 7. Shoot (A) and root (B) GR activity in A. maritimus plants. See legend for Fig. 1. (nd means non-
determined enzyme activity).

A
60 a
Ri collect
Ri CdG A
50 Sc CdG
Ce CdG G
40 b D G
APX activity (nmol Asc min-1mg-1prot)

30 a a
J
20 E b
10 B E H K c K
a a a a
nd

c
0
0 mM 100 mM 175 mM

G
400 a
A B
350 H H
a J
300 B a ab D J
D ab
250 a b b
b
200 E K
150 b b
100
50
nd

0
0 mM 100 mM 175 mM

Fig. 8. Shoot (A) and root (B) APX activity in A. maritimus plants. See legend for Fig. 1. (nd means non-
determined enzyme activity).

197
Chapter 6

Leaf APX activity increased considerably at 100 mM NaCl in plants inoculated


with Ri collect, as well as, in plants inoculated with Sc CdG (Fig. 8A). In contrast,
plants inoculated with Ri CdG and Ce CdG only increased their leaf APX activity at
175 mM NaCl. In roots, under non-saline conditions, plants inoculated with Ri collect
and Sc CdG had the highest levels of APX activity (Fig. 8B). APX activity also
increased with salt increase, but the increase was slight at 100 mM NaCl. At 175 mM
NaCl plants inoculated with Sc CdG showed the highest root APX activity (Fig. 8B).

Discussion

A. maritimus, a native halophyte plant from Mediterranean areas, showed a high


degree of mycotrophy since all non-mycorrhizal plants did not survive, even in absence
of an additional NaCl treatment. This evidences the importance of the rhizosphere
microbiota, particularly the AMF, in the establishment and growth of this plant species.
Herrera et al. (1993) showed that the restoration of a desertified area was most
successful using native plant species inoculated with soil microbiota, including AMF. In
fact, Klironomos (2003) concluded that for the establishment of a diverse plant
community it is important to use locally adapted AMF community, as plant species
benefited most from certain AMF species. In fact, at the highest salinity level, only 30%
of A. maritimus plants inoculated with the collection AMF survived, while with the
three native AMF, the rate of survival was 100%, pointing out the importance of
inoculation with native-AMF in the survival of this plant species. Thus, any attempt of
revegetation of degraded saline Mediterranean areas using A. maritimus should consider
the mycorrhizal dependency of this plant and the use of adequate inocula. Although
there are AMF species which seem to be globally distributed (Öpik et al. 2006), van der
Heijden et al. (1998) pointed out that plant species benefited differently from the fungal
strains regarding biomass production. Our results showed that plants inoculated with
native AMF had higher shoot dry weight than those inoculated with the collection AMF
at the highest level of salinity. This is in accordance to Requena et al. (2001) who found
that native AMF species were more efficient in promoting plant growth of Anthyllis
cytisoides than AMF derived from a different habitat. Similar results were obtained in
Pulsatilla species (Moora et al. 2004), Conyza bilbaoana (Oliveira et al. 2005) and
Arnica montana (Vergeer et al. 2006).
The nature of AMF hampers the progress in mycorrhiza research, especially in
the field of applied ecology (Rosendahl 2008; Young 2008). Thus in this work we tried
to elucidate differences in symbiotic efficiency among three AMF native from a saline
Mediterranean area and a collection AM fungus when associated to A. maritimus.
Mycorrhizal colonization was not affected by increasing salinity, as some other authors
found (Yamato et al. 2008; Wu et al. 2010). Although Ri collect had the highest
percentage of root colonization, the native AMF strains maintained a higher symbiotic

198
Chapter 6

efficiency with A. maritimus, as well as a 100% of survival rate under 175 mM NaCl. In
fact, the high percentage of root colonization by Ri collect could demand excessive
carbohydrates from the plant (Klironomos 2003).
In this study, when plants of A. maritimus were subjected to high salinity stress
(175 mM NaCl), plants inoculated with the three native-AMF exhibited better
performance of photosystem II and higher stomatal conductance. Results on efficiency
of photosystem II and stomatal conductance indicate that plants inoculated with native-
AMF may improve the net assimilation rates by protecting the photosynthetic
machinery and enhancing transpiration rates. Some other authors showed a similar
tendency (Sheng et al. 2008; Hajiboland et al. 2010) and Estrada et al. (2012) reported
that the improvement in photosystem II was higher with native AMF than with
collection fungi together with the enhancement of plant stomatal conductance, in
agreement with Querejeta et al. (2006). These two effects may have accounted for the
enhanced growth of A. maritimus during high salinity stress.
In addition to the beneficial effect of native mycorrhizal colonization in A.
maritimus salt tolerance by improving photosynthetic ability and stomatal conductance;
water and nutrient uptake, ion balance and osmolite concentration among others may
have also contributed to it (Ruiz-Lozano et al. 2012). It has been previously shown that
a native AMF from Cabo de Gata developed better under salinity than a collection
fungal strain (Estrada et al., 2012). Thus, the extensive hyphal network of native AMF
can explore a larger soil volume, contributing to water and nutrient uptake (Evelin et al.
2012). The maintenance of proper ionic homoeostasis and osmotic adjustment may also
prevent salt injury. The synthesis of organic osmolites, especially proline that is
normally located in the cytosol, appears to be of importance in osmotic adjustment
(Moghaieb et al. 2004). Our results did not show remarkable differences in proline
content among fungal treatments, although proline accumulation increased in all
treatments with the salinity stress. The latter may be explained as halophytic plants are
themselves tolerant to salinity in part because they are able to maintain a high osmotic
potential through the accumulation of organic solutes (Bradley and Morris 1991).
Salinity stress leads to secondary oxidative damage, thus the improvement of
stress tolerance is often related to enhancement of contents of antioxidant compounds in
plants. Due to the toxicity of ROS, plants have developed appropriate detoxification
systems to remove these molecules. These systems include, among others, non-
enzymatic antioxidants soluble compounds, such as glutathione and ascorbate that are
major plant metabolites that regulate essential cell functions and play a key role in
antioxidant defence (Tunc-Ozdemir et al. 2009). Glutathione reacts with superoxide
radicals, peroxy radicals and singlet oxygen and forms oxidized glutathione and other
disulphides (Meyer 2007). The ascorbate is involved in the removal of H2O2 by
ascorbate peroxidases, which use ascorbate as electron donor, and is closely related to
glutathione in the ascorbate-glutathione cycle where glutathione participates in the
reduction of oxidized ascorbate (Noctor and Foyer 1998). Our results showed that

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Chapter 6

glutathione content had the most significant differences among treatments. A. maritimus
plants inoculated with native AMF increased it shoot glutathione content with
increasing salinity. Plants inoculated with Ri collect reduced the content at 175 mM
NaCl and at this salinity level, the content of glutathione in shoots was lower compared
to native AMF-inoculated plants. In contrast, the ascorbate content did not show a
significant trend. From these results we may propose that A. maritimus has preference
for glutathione as an antioxidant compound compared to ascorbate. The reason for that
could be related to the additional physiological functions ascribed to glutathione, such
as the induction of enzyme activities and its participation in sulphur metabolism and
regulation of gene expression (Foyer et al. 1995).
Another major effect of salinity stress in plants is the loss of membrane integrity
due to the oxidation of membrane lipids. Thus the prevention of lipid peroxidation is of
crucial importance to maintain membrane integrity (Garg and Manchanda 2009). Our
findings showed that in A. maritimus, MDA content (a specific product of lipid
peroxidation induced by ROS) increased with salinity in shoots but not in roots, with
exception of Sc CdG inoculated plants, which always increased the MDA content. Lipid
peroxidation is considered an useful indicator of cellular oxidative damage (Li et al.
2012), however it might not be a good indicator in halophytes due to the particular
characteristics of these plants. We cannot compare with some other works that observed
a reduction of oxidative damage to lipids by AM symbiosis in plants subjected to salt
stress (He et al. 2007; Hajiboland et al. 2010) because of the lack of non-mycorrhizal
plants. In spite of that, we observed that, at each salt level, plants inoculated with Sc
CdG always had the lowest MDA content.
Oxidative damage in plants can be also ameliorated by an efficient antioxidative
enzyme system that may confer plants the ability to tolerate salt stress (Mittler 2002).
The induction of ROS-scavenging enzymes, such as SOD, GR and APX is the most
common mechanism for detoxifying ROS synthesized during stress response
(Hajiboland et al. 2010). In halophytes the effects of AMF on the activities of
antioxidant enzymes have been rarely studied (Li et al. 2012). Data from this study is
difficult to discuss because there are no data with respect of Ri collect inoculated plants
at 175 mM NaCl. Despite of all the above mentioned, the activity of SOD in shoots only
increased in plants inoculated with Sc CdG and Ce CdG. In roots, plants inoculated with
Sc CdG showed the most important increase in SOD activity, having the highest activity
both in shoots and in roots. This may have accounted for the lower MDA content in A.
maritimus plants inoculated with Sc CdG compared to the rest of the treatments.
Although the pattern of GR activity in roots was not relevant, in shoots of A.
maritimus it was higher in plants inoculated with the three native AMF at the highest
level of salinity. The enhanced GR activity in native AMF inoculated plants may have
contributed to generate reduced glutathione that plays a pivotal role in the ascorbate-
glutathione cycle among others. As for the APX activity, the data showed that it was
enhanced by the increase in salinity in all treatments both in shoots and in roots. In

200
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accordance to He et al. (2007), our results indicated that AMF induce APX activity.
Nonetheless, native AMF did not enhance the activity more than the Ri collect fungus
contrary to the other studied enzymes. It is known that the response of each enzyme
differs with respect not only to the fungal species but to the host plant (Roldán et al.
2008; Evelin et al. 2009; Ruiz-Lozano et al. 2012).
Summarizing, the present study showed the elevated degree of mycotrophy of A.
maritimus plants, especially regarding native AMF from its environment. In addition,
we have observed several physiological mechanisms by which native AMF ameliorate
the detrimental effects of salinity stress in A. maritimus better than an exotic AMF, such
as better photosynthetic efficiency or higher levels of glutathione and of certain
antioxidant enzyme activities. In degraded semiarid Mediterranean areas, Alguacil et al.
(2011) demonstrated the important impact of inoculation with native AM fungi on the
growth of shrub species. Moreover, reestablishment of plants species and restoration of
specific ecosystems need careful considerations regarding the AMF inocula and the
plant species used. Also the introduction of novel AMF species or isolates to the site
could result in the out-competition of native fungal strains that could provoke the up-
coming or survival of invasive plant species (Schwartz et al. 2006). Thus, inadequate
inocula should be avoided due to possible negative effects on plant species and the plant
community. Van der Heijden et al. (1998) showed that under particular environmental
conditions, different AMF strains had negative or positive effect on the colonized plant,
pointing out the importance of the interactions between AMF and plant species. The
results in the present work remark the importance of native AMF inoculation in A.
maritimus establishment, survival and growth for successful restoration of
Mediterranean areas, particularly on sites with high salt levels in the soil.

Acknowledgements

This work was financed by two research projects supported by Junta de


Andalucía (Spain). Projects P06-CVI-01876 and P11-CVI-7107. We thank Sonia
Molina for technical assistance and Domingo Álvarez (curator of the EEZ germplasm
collection), for taking care of the native AMF inocula.

201
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References

Alguacil MM, Torres MP, Torrecillas E, Díaz G, Roldán A (2011) Plant type differently
promote the arbuscular mycorrhizal fungi biodiversity in the rhizosphere after
revegetation of a degraded, semiarid land. Soil Biol Biochem 43:167-173.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment
search tool. J Mol Biol 215:403-410
Aroca R, Irigoyen JJ, Sánchez-Díaz M (2001) Photosynthetic characteristics and
protective mechanisms against oxidative stress during chilling and subsequent
recovery in two maize varieties differing in chilling sensitivity. Plant Sci
161:719-726
Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-
stress studies. Plant Soil 39:205-207
Becana M, Aparicio-Tejo P, Irigoyen JJ, Sánchez-Díaz M (1986) Some enzymes of
hydrogen peroxide metabolism in leaves and root nodules of Medicago sativa.
Plant Physiol 82:1169-1171
Becker WN, Gerdemann JW (1977) Glomus etunicatus sp. nov. Mycotaxon 6:29-32
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification.
Can J Biochem Physiol 37:911-917
Bradford MM (1976) A rapid and sensitive method for quantitation of microgram
quantities of protein utilizing principle of protein-dye binding. Anal Biochem
72:248-254
Bradley PM, Morris JT (1991) Relative importance of ion exclusion, secretion and
accumulation in Spartina alterniflora Loisel. J Exp Bot 42:1525-1532
Brito I, de Carvalho M, Goss MJ (2011) Summer survival of arbuscular mycorrhiza
extraradical mycelium and the potential for its management through tillage
options in Mediterranean cropping systems. Soil Use Manage 27:350-356.
Brundrett M, Melville L, Peterson L (1994) Practical methods in mycorrhizal research.
Mycologue Publications, University of Guelph, Ontario, Canada
Ding M, Hou P, Shen X, Wang M, Deng S, Sun J, Xiao F, Wang R, Zhou X, Lu C,
Zhang D, Zheng X, Hu Z, Chen S (2010) Salt-induced expression of genes
related to Na+/K+ and ROS homeostasis in leaves of salt-resistant and salt-
sensitive poplar species. Plant Mol Biol 73:251-269
Enkhtuya B, Rydlova J, Vosátka M (2000) Effectiveness of indigenous and non-
indigenous isolates of arbuscular mycorrhizal fungi in soils from degraded
ecosystems and man-made habitats. Appl Soil Ecol 14:201-211
Estrada B, Barea JM, Aroca R, Ruiz-Lozano JM (2012) A native Glomus intraradices
strain from a Mediterranean saline area exhibits salt tolerance and enhanced
symbiotic efficiency with maize plants under salt stress conditions. Plant and
Soil (In press). doi:10.1007/s11104-012-1409-y

202
Chapter 6

Evelin H, Giri B, Kapoor R (2012) Contribution of Glomus intraradices inoculation to


nutrient acquisition and mitigation of ionic imbalance in NaCl-stressed
Trigonella foenum-graecum. Mycorrhiza 22:203-217
Evelin H, Kapoor R, Giri B (2009) Arbuscular mycorrhizal fungi in alleviation of salt
stress: a review. Ann Bot 104:1263-1280.
Ferrol N, Calvente R, Cano C, Barea JM, Azcón-Aguilar C (2004) Analysing arbuscular
mycorrhizal fungal diversity in shrub-associated resource islands from a
desertification-threatened semiarid Mediterranean ecosystem. Appl Soil Ecol
25:123-133.
Foyer CH, Souriau N, Perret S, Lelandais M, Kunert KJ, Pruvost C, Jouanin L (1995)
Overexpression of glutathione-reductase but not glutathione synthetase leads to
increases in antioxidant capacity and resistance to photoinhibition in poplar
trees. Plant Physiol 109:1047-1057
Garg N, Manchanda G (2009) Role of arbuscular mycorrhizae in the alleviation of
ionic, osmotic and oxidative stresses induced by salinity in Cajanus cajan (L.)
Millsp (pigeonpea). J Agron Crop Sci 195:110-123.
Geiger F (1973) El Sureste español y los problemas de la aridez. Revista de geografia
7:166-209
Giovannetti M, Mosse B (1980) Evaluation of techniques for measuring vesicular
arbuscular mycorrhizal infection in roots. New Phytol 84:489-500
Hajiboland R, Aliasgharzadeh N, Laiegh SF, Poschenrieder C (2010) Colonization with
arbuscular mycorrhizal fungi improves salinity tolerance of tomato (Solanum
lycopersicum L.) plants. Plant Soil 331:313-327.
Halliwell B, Gutteridge JMC (1989) Free radicals in biology and medicine. Clanderon
Press, Oxford, UK
He Z, He C, Zhang Z, Zou Z, Wang H (2007) Changes of antioxidative enzymes and
cell membrane osmosis in tomato colonized by arbuscular mycorrhizae under
NaCl stress. Colloids Surf B 59:128-133.
Herrera MA, Salamanca CP, Barea JM (1993) Inoculation of woody legumes with
selected arbuscular mycorrhizal fungi and rhizobia to recover desertified
mediterranean ecosystems. Appl Environ Microbiol 59:129-133
Jeffries P, Barea JM (2012) Arbuscular Mycorrhiza - a key component of sustainable
plant-soil ecosystems. In: Hock B (ed) The Mycota, vol IX. Fungal
Associations, 2nd edition. Springer-Verlag, Berlin, Heidelberg, pp 95-113
Jeffries P, Craven-Griffiths A, Barea JM, Levy Y, Dodd JC (2002) Application of
arbuscular mycorrhizal fungi in the revegetation of desertified Mediterranean
ecosystems. In: Gianinazzi S, Schüepp H, Barea JM, Haselwandter K (eds)
Mycorrhiza Technology in Agriculture: from Genes to Bioproducts Birkhäuser
Verlag,, Basel, Switzerland, pp 151-174
Juniper S, Abbott L (2006) Soil salinity delays germination and limits growth of hyphae
from propagules of arbuscular mycorrhizal fungi. Mycorrhiza 16:371-379.

203
Chapter 6

Klironomos JN (2003) Variation in plant response to native and exotic arbuscular


mycorrhizal fungi. Ecology 84:2292-2301
Koske RE, Tessier B (1983) A convenient, permanent slide mounting medium. Myc
Soc Am Newsl 34:59
Krüger M, Krüger C, Walker C, Stockinger H, Schussler A (2012) Phylogenetic
reference data for systematics and phylotaxonomy of arbuscular mycorrhizal
fungi from phylum to species level. New Phytol 193:970-984
Lee J, Lee S, Young JPW (2008) Improved PCR primers for the detection and
identification of arbuscular mycorrhizal fungi. FEMS Microbiol Ecol 65:339-
349.
Leipner J, Fracheboud Y, Stamp P (1997) Acclimation by suboptimal growth
temperature diminishes photooxidative damage in maize leaves. Plant Cell
Environ 20:366-372
Lendínez ML, Marchal FM, Salazar C (2011) Estufio florístico de los medios húmedos
salinos de Andalucía (S. España). Catálogo y análisis de la flora vascular
halófila. Lagascalia 31:77-130
Li T, Liu RJ, He XH, Wang BS (2012) Enhancement of superoxide dismutase and
catalase activities and salt tolerance of euhalophyte Suaeda salsa L. by
mycorrhizal fungus Glomus mosseae. Pedosphere 22:217-224
Mason E (1928) Note on the presence of mycorrhizae in the roots of salt marsh plants.
New Phytol 27:193-195
Meyer AJ (2007) The integration of glutathione homeostasis and redox signaling. J
Plant Physiol 31:1-14
Minotti G, Aust SD (1987) The requirement for iron (III) in the initiation of lipid-
peroxidation by iron(II) and hydrogen-peroxide. J Biol Chem 262 (3):1098-1104
Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci
7:405-410
Moghaieb REA, Saneoka H, Fujita K (2004) Effect of salinity on osmotic adjustment,
glycinebetaine accumulation and the betaine aldehyde dehydrogenase gene
expression in two halophytic plants, Salicornia europaea and Suaeda maritima.
Plant Sci 166:1345-1349
Moora M, Öpik M, Sen R, Zobel M (2004) Native arbuscular mycorrhizal fungal
communities differentially influence the seedling performance of rare and
common Pulsatilla species. Funct Ecol 18:554-562
Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol
59:651-681
Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific
peroxidase in spinach chloroplast. Plant Cell Physiol 22:867-880
Noctor G, Foyer CH (1998) Ascorbate and glutathione: Keeping active oxygen under
control. Annu Rev Plant Physiol Plant Mol Biol 49:249-279

204
Chapter 6

Oehl F, Sieverding E, Palenzuela J, Ineichen K, Silva GA (2011) Advances in


Glomeromycota taxonomy and classification. IMA Fungus 2:191-199
Oliveira RS, Vosátka M, Dodd JC, Castro PML (2005) Studies on the diversity of
arbuscular mycorrhizal fungi and the efficacy of two native isolates in a highly
alkaline anthropogenic sediment. Mycorrhiza 16:23-31
Öpik M, Moora M, Liira J, Zobel M (2006) Composition of root-colonizing arbuscular
mycorrhizal fungal communities in different ecosystems around the globe. J
Ecol 94:778-790
Oxborough K, Baker NR (1997) Resolving chlorophyll a fluorescence images of
photosynthetic efficiency into photochemical and non-photochemical
components - calculation of qP and Fv '/Fm ' without measuring Fo '. Photosynth
Res 54:135-142.
Phillips JM, Hayman DS (1970) Improved procedure of clearing roots and staining
parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of
infection. Trans Br Mycol Soc 55:159-161
Querejeta JI, Allen MF, Caravaca F, Roldán A (2006) Differential modulation of host
plant δ13C and δ18O by native and nonnative arbuscular mycorrhizal fungi in a
semiarid environment. New Phytol 169:379-387.
Requena N, Jeffries P, Barea JM (1996) Assessment of natural mycorrhizal potential in
a desertified semiarid ecosystem. Appl Environ Microbiol 62:842-847
Requena N, Pérez-Solis E, Azcón-Aguilar C, Jeffries P, Barea JM (2001) Management
of indigenous plant-microbe symbioses aids restoration of desertified
ecosystems. Appl Environ Microbiol 67:495-498
Rodríguez P, Torrecillas A, Morales MA, Ortuño MF, Sánchez-Blanco MJ (2005)
Effects of NaCl salinity and water stress on growth and leaf water relations of
Asteriscus maritimus plants. Environ Exp Bot 53:113-123
Rodriguez R, Redman R (2008) More than 400 million years of evolution and some
plants still can’t make it on their own: plant stress tolerance via fungal
symbiosis. J Exp Bot 59:1109-1114
Roldán A, Díaz-Vivancos P, Hernández JA, Carrasco L, Caravaca F (2008) Superoxide
dismutase and total peroxidase activities in relation to drought recovery
performance of mycorrhizal shrub seedlings grown in an amended semiarid soil.
J Plant Physiol 165:715-722.
Rosendahl S (2008) Communities, populations and individuals of arbuscular
mycorrhizal fungi. New Phytol 178:253-266.
Ruiz-Lozano JM, Porcel R, Azcón R, Aroca R (2012) Regulation by arbuscular
mycorrhizae of the integrated physiological response to salinity in plants: new
challenges in physiological and molecular studies. J Exp Bot 63:4033-4044.
Schaedle MY, Bassham JA (1977) Chloroplast glutathione reductase. Plant Physiol
59:1011-1012

205
Chapter 6

Schenk NC, Smith GS (1982) Additional new and unreported species of mycorrhizal
fungi (Endogonaceae) from Florida. Mycologia 74:77-92
Schwartz MW, Hoeksema JD, Gehring CA, Johnson NC, Klironomos JN, Abbott LK,
Pringle A (2006) The promise and the potential consequences of the global
transport of mycorrhizal fungal inoculum. Ecol Lett 9:501-515
Sheng M, Tang M, Chen H, Yang B, Zhang F, Huang Y (2008) Influence of arbuscular
mycorrhizae on photosynthesis and water status of maize plants under salt stress.
Mycorrhiza 18:287-296.
Sieverding E (1991) Vesicular-arbuscular mycorrhiza management in tropical
agrosystems. TZ-Verlagsgesellschaft, Technical Cooperation (GTZ), Eschborn,
Friedland, Bremer, Rossdorf, Germany
Smith IK (1985) Stimulation of glutathione synthesis in photorespiring plants by
catalase inhibitors. Plant Physiol 79:1044-1047
Smith SE, Read DJ (2008) Mycorrhizal Symbiosis, 3rd Ed. Elsevier, Academic Press,
New York
Sonjak S, Udovic M, Wraber T, Likar M, Regvar M (2009) Diversity of halophytes and
identification of arbuscular mycorrhizal fungi colonising their roots in an
abandoned and sustained part of Secovlje salterns. Soil Biol Biochem 41:1847-
1856.
Spain JL (1990) Arguments for diagnoses based on unaltered wall structures.
Mycotaxon 38:71-76
Tawfik MM, Thalooth AT, Zaki NM (2010) Sustainable restoration of salt-affected soil
through revegetation of Leptochloa fusca and Sporobolus virginicus. In: Thomas
RP (ed) Proceedings of the Global Forum on Salinization and Climate Change
(GFSCC2010). FAO, Rome, Italy
Trappe JM (1977) Three new Endogonaceae: Glomus constrictus, Sclerocystis
clavispora and Acaulospora scrobiculata. Mycotaxon 6:359-366
Tunc-Ozdemir M, Miller G, Song L, Kim J, Sodek A, Koussevitzky S, Misra AN,
Mittler R, Shintani D (2009) Thiamin confers enhanced tolerance to oxidative
stress in Arabidopsis. Plant Physiol 151:421-432
Valladares F (2004) Global change and radiation in Mediterranean forest ecosystems: a
meeting point for ecology and management. In: Arianoutsou M, Papanastasis, V.
(ed) Ecology, conservation and sustainable management of Mediterranean type
ecosystems of the world. Millpress, Rotterdam, the Netherlands. pp 1-4
Vallejo VR, Aronson J, Pausas JG, Cortina J (2005) Restoration of Mediterranean
woodlands. In: Andel JV, Aronson JJ (eds) Restoration ecology: the new
frontier. Blackwell Publishing, Oxford, United Kingdom, pp 193-207
van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R,
Boller T, Wiemken A, Sanders IR (1998) Mycorrhizal fungal diversity
determines plant biodiversity, ecosystem variability and productivity. Nature
396:69-72

206
Chapter 6

Vergeer P, van den Berg LJL, Baar J, Ouborg NJ, Roelofs JGM (2006) The effect of
turf cutting on plant and arbuscular mycorrhizal spore recolonisation:
implications for heathland restoration. Biol Conserv 129:226-235
Wilde P, Manal A, Stodden M, Sieverding E, Hildebrandt U, Bothe H (2009)
Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt
marshes. Environ Microbiol 11:1548-1561.
Wu QS, Zou YN, He XH (2010) Contributions of arbuscular mycorrhizal fungi to
growth, photosynthesis, root morphology and ionic balance of citrus seedlings
under salt stress. Acta Physiol Plant 32:297-304.
Yamato M, Ikeda S, Iwase K (2008) Community of arbuscular mycorrhizal fungi in a
coastal vegetation on Okinawa island and effect of the isolated fungi on growth
of sorghum under salt-treated conditions. Mycorrhiza 18:241-249.
Young JPW (2008) The genetic diversity of intraterrestrial aliens. New Phytol 178:465-
468
Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6:66-71

207
CONCLUSIONES
CONCLUSIONS
Conclusiones

CONCLUSIONES

1. Las micorrizas arbusculares se encuentran ampliamente distribuidas en


ambientes salinos, como son las dunas y las marismas del Parque Natural de
Cabo de Gata. Este estudio ha permitido describir un total de 30 morfotipos de
esporas pertenecientes a tres clases, cinco órdenes, nueve familias y trece
géneros. A pesar de que la diversidad fue bastante similar en ambos sitios, en la
rizosfera de la marisma la densidad de esporas fue seis veces mayor que en la
duna.

2. En las dunas del Parque Natural de Cabo de Gata se ha descrito una nueva
especie de MA asociada a la rizosfera de Asteriscus maritimus y existe la
posibilidad de encontrar más especies aún desconocidas.

3. El aislado nativo de Cabo de Gata Glomus (= Rhizophagus) intraradices ha


demostrado tener una mayor capacidad de desarrollo en medios salinos que un
aislado de colección de la misma especie, indicando mayor adaptación a la
salinidad que el hongo de colección. Este efecto puede estar relacionado con la
considerable mayor expresión de los genes GintBIP, Gint14-3-3 y GintAQP1, en
el hongo aislado de Cabo de Gata.

4. En condiciones de salinidad, el aislado nativo de Cabo de Gata G. intraradices


presenta además una mayor eficiencia simbiótica con plantas de maíz que un
aislado de la misma especie de colección, promoviendo un mayor desarrollo de
las plantas.

5. El uso de hongos MA nativos aislados de Cabo de Gata incrementó la tolerancia


a la salinidad de una planta glicófita de interés agronómico como es el maíz, en
mayor medida que un hongo MA de colección. Las plantas inoculadas con los
tres hongos nativos mostraron un aumento significativo de la relación K+/Na+ en
sus tejidos. Este efecto se correlacionó con la regulación de la expresión de los
genes ZmAKT2, ZmSOS1 y ZmSKOR en las raíces de dichas plantas,
contribuyendo a una mejor homeostasis iónica.

6. Las plantas de maíz inoculadas con los tres hongos nativos de Cabo de Gata
mostraron mayor eficiencia del fotosistema II y conductancia estomática, lo que
habría contribuido a reducir la fotorrespiración y la producción de ROS.
Además, los sistemas antioxidantes de estas plantas fueron inducidos en mayor
medida que en las plantas no micorrizadas o en las inoculadas con el hongo MA
de colección, siendo el daño oxidativo significativamente menor.

211
Conclusiones

7. La planta halófita Asteriscus maritimus, de interés en programas de revegetación


de ecosistemas salinos degradados, ha demostrado un elevado grado de
micotrofía y ser dependiente de hongos MA nativos para su completa
supervivencia y desarrollo en condiciones de salinidad elevada.

8. Existen diferencias en los mecanismos por los que los hongos MA nativos de
Cabo de Gata incrementan la tolerancia de la planta hospedadora a la salinidad.
Esto estaría modulado, igualmente, por las características fisiológicas de la
especie de planta utilizada.

212
Conclusions

CONCLUSIONS

1. Arbuscular mycorrhiza fungi are widely distributed in saline environments, such


as dunes and salt marshes of the Natural Park of Cabo de Gata. This study
describes a total of 30 spore morphotypes belonging to three classes, five orders,
nine families and thirteen genera. Although diversity was similar at both sites, in
the rhizosphere of the salt marsh spore density was six times higher than in the
dune.

2. A new species of AMF associated with the rhizosphere of Asteriscus maritimus


was described in the dunes of the Natural Park of Cabo de Gata. There is a
possibility to find more still undescribed species.

3. The native isolate from Cabo de Gata Glomus (= Rhizophagus) intraradices has
shown to have greater development capacity under saline conditions than an
isolate of the same species from collection, indicating higher adaptation to
salinity than the fungus from collection. This effect may be related to the
significant up-regulation in the expression of the genes GintBIP, Gint14-3-3 and
GintAQP1, in the fungus isolated from Cabo de Gata.

4. Under saline conditions, the native isolate from Cabo de Gata G. intraradices
had also higher symbiotic efficiency with maize plants than an isolate of the
same species from collection, promoting a greater development of the plants.

5. The use of native AM fungi isolated from Cabo de Gata increased the salt
tolerance of a glycophyte plant of agronomic interest, such as maize, more than
a fungus from collection. Plants inoculated with the three native fungi showed a
significant increase of the K+/Na+ ratios in their tissues. This effect was
correlated with the regulation of expression of ZmAKT2, ZmSKOR and ZmSOS1
genes in the roots of such plants, contributing to improve the ionic homeostasis.

6. Maize plants inoculated with the three native AM fungi from Cabo de Gata
showed higher efficiency of photosystem II and stomatal conductance, which
should have contributed to reduce photorespiration and ROS production. In
addition, the antioxidant systems of these plants were induced to a greater extent
than in non-mycorrhizal plants or plants inoculated with the fungus from
collection, and they had a significantly lower oxidative damage.

213
Conclusions

7. The halophyte Asteriscus maritimus, a plant of interest in revegetation of saline


degraded ecosystems, has shown to have a high degree of micotrophy and to be
dependent of native AM fungi for its complete survival and development under
high salinity conditions.

8. There are differences in the mechanisms by which native AMF of Cabo de Gata
increase host plant tolerance to salinity. This would be also modulated by the
particular physiological characteristics of the plant species used.

214

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